- Email: [email protected]
Above- and below-ground responses of Eragrostis and Bouteloua grass seedlings to the plant-growth-promoting bacterium Azospirillium brasilense
ARTICLE IN PRESS Journal of Arid Environments Journal of Arid Environments 59 (2004) 19–26 www.elsevier.com/locate/jnlabr/yjare
Above- and below-ground responses of Eragrostis and Bouteloua grass seedlings to the plant-growth-promoting bacterium Azospirillium brasilense He! ctor O. Rubio Ariasa, M. Karl Woodb,*, Carlos c ! Morales Nietoc, Gerardo Reyes Lopez , Lourdes de la Vegad a
Campo Experimental Madera, Instituto Nacional de Investigaciones Forestales, Agr!ıcolas y Pecuarias-SAGARPA, Ave. Homero No. 3744, Chihuahua, Mexico b Department of Animal and Range Science, New Mexico State University, Box 3-I, Las Cruces, NM 88003, USA c Campo Experimental la Campana, INIFAP-SAGARPA, Ave. Homero 3744, Chihuahua, Chihuahua C.P. 31100, Mexico d ! ! Facultad de Zootecnia de la Universidad Autonoma de Chihuahua, Kilometro 7.5 Carr. Chihuahua, Cuauht!emoc, Chihuahua, Mexico Received 27 January 2003; received in revised form 3 November 2003; accepted 14 January 2004
Abstract Two experiments were performed to determine the effects of inoculation with N2 fixing bacterium Azospirillum brasilense on forage production and root growth in seedlings of Wilman lovegrass, weeping lovegrass, and sideoats grama. Two sources of inorganic nitrogen (N) were tested: Urea, CO(NH2)2 (45%) and ammonium nitrate NH4NO3 (33.3%). Crude protein (CP), calcium (Ca) and phosphorous (P) concentrations were determined. In both experiments top dry matter (DM) production was significantly affected for grasses and inoculation but no differences were found for inorganic nitrogen applications. Maximum amount of top DM was obtained with the Wilman lovegrass with 5.70 g, weeping lovegrass reached 2.70 g, and sideoats grama only reached 1.58 g. Inoculated treatments reached 3.37 g while the no-inoculated treatments obtained 2.74 g. A similar trend was also noted for root phytomass. CP, Ca and P concentrations for the first experiment did not show any trend; therefore, they were not determined for the second experiment. Inoculation of grasses with
*Corresponding author. E-mail address: [email protected] (M.K. Wood). 0140-1963/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2004.01.008
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A. brasilense may be a feasible practice for seeding some rangeland where N deficiency is a problem. r 2004 Elsevier Ltd. All rights reserved. Keywords: Nitrogen fixing bacteria; Azospirillium; Inorganic nitrogen; Root phytomass; Above-ground production
1. Introduction and literature Two decades ago, 40% of Mexico’s land was classified as arid or semi-arid (Molina, 1983) although more recent estimations consider that percentage to be as high as 52% (CONAZA, 1994). Across the world, arid and semi-arid land comprises about 40%, and it is believed that it is growing larger (Hayes, 1991). In addition, it is estimated that 6.7% of the Earth’s land has been turned into deserts; hence, it is expected that arid and semi-arid land will increase in Mexico as well as all over the world. Soil orders frequently found in arid and semi-arid environments include Aridisols, Entisols, Oxisols and Vertisols (Flach and Smith, 1969), which are low in organic matter, and as a consequence, low in nitrogen (N). It has been suggested for diverse ecological systems that the accumulation, degradation, and utilization of poly(bhydroxybutyrate) by several bacteria under stress is a mechanism that favors their establishment, proliferation, survival, and competition, especially in competitive environments where carbon and energy sources are limiting, such as those encountered in the soil and rhizosphere (Kadouri et al., 2002; Okon and Itzigsohn, 1992). In Mexico, a study showed that 70% of all soils have less than 1% organic matter (Estrada, 1987); therefore, the main problem in arid areas, without considering water supply is low levels of N. Although the atmosphere is 78% N, this element cannot be used by plants (Donahue et al., 1983) with the exception of two cases: N fixing by the symbiosis of the complex Leguminosae–Rhizobium that depends on formation of nodules in roots and for some N fixing bacteria that have the ability to fix N without nodulation. Recent information showed that world production of N fertilizer has overtaken the other sources of supply to plants (Jenkinson, 2001). Nitrogen fixing bacteria have been broadly studied because of their biological as well as their economic importance in areas such as medical use, degradation of pollutants, vitamin production, and purification of residual water (Bashan and Holguin, 1997). Few studies has been conducted on perennial grasses (Eckert et al., 2001) and even fewer by inoculating perennial plant species found in rangeland areas. The genera Azospirillum, Azotobacter, and Clostridiumn belong to the Azotobacteraceae family, which are bacteria that fix nitrogen without forming nodules in the plants’ roots. Azospirillum is aerobic and possesses a metabolism where the O2 or the NO3 are the electron acceptors (Haurat et al., 1994). Presently, there are five different species: Azospirillum brasilense, A. lipoforum, A. amazonense, A. halopraeferens, and A. irakense (Tarrand et al., 1978; Khammas et al., 1989;
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Givaudan and Bally, 1991). Bouton and Zuberer (1979) inoculated the bacteria A. brasilense to the grass Panicum maximum and noted an increase in biomass production. They also found that inoculation increased nitrogen content in the plant tissue. Other research conducted in Israel (Avivi and Feldman, 1982; Inbal and Feldman, 1982), Egypt (Hegazi et al., 1981; Hegazi et al., 1983), and Brazil (Boddey et al., 1986) obtained similar results. Moreover, some researchers have suggested that certain bacteria, such as Azospirillum, might provide higher yields in cereals (Smith et al., 1984) as well as in some non-cereal crop plants (Bashan et al., 1989; Zaady et al., 1994). The above-mentioned effects of bacteria inoculation can result in more root volume (Barbieri et al., 1986), an increase in root biomass (Hadas and Okon, 1987) and enlarged and healthier root systems (Venkateswarlu and Rao, 1983). The largest portion of Azospirillum literature consists of genetic studies of almost all aspects of the bacterium and its association with plants (Bashan and Holguin, 1997). To date little information is available on the effects of inoculating free living bacteria on non-cereal and perennial grass species’ yields (Bashan and Holguin, 1997); therefore, the objective of this research was to determine the effects of inoculating A. brasilense on the seeds of Wilman lovegrass (Eragrostis superba Peyn.), weeping lovegrass (E. curvula [Schrad.] Nees) and sideoats grama (Bouteloua curtipendula [Mich.] Torr.) with and without applying inorganic nitrogen fertilizer. These results are of practical importance to northern Mexico because these grass species are recommended for seeding rangelands.
2. Materials and methods Two experiments were conducted in the greenhouse of the Faculty of Zootechnic of the Autonomus University of Chihuahua, Mexico, during 1993 and 1994. The soil was collected in an undisturbed area and analysed in the Soil and Water Testing Lab at New Mexico State University in October 18, 1993. Experiments were conducted in 48 containers (19.5 cm diameter, 31.70 cm height) containing 8 kg of dry soil. In both experiments, the seeds of the grasses to be tested were inoculated with the bacteria A. brasilense. The first experiment evaluated application of urea CO(NH2)2 (45%), while in the second experiment the application of ammonium nitrate NH4NO3 (33.3%) as inorganic nitrogen was evaluated. A 3 2 2 factorial arrangement of treatments (three grass species, two levels of inoculation and two levels of inorganic nitrogen application) was employed. Hence, treatments were the followings: (i) Wilman lovegrass–inoculated–fertilizer; (ii) Wilman lovegrass–inoculated–no fertilizer; (iii) Wilman lovegrass–no inoculation–fertilizer; (iv) Wilman lovegrass– inoculation–no fertilizer; (v) weeping lovegrass–inoculated–fertilizer; (vi) weeping lovegrass–inoculated–no fertilizer; (vii) weeping lovegrass–no inoculation–fertilizer; (viii) weeping lovegrass–no inoculation–no fertilizer; (ix) sideoats grama–inoculated– fertilizer; (x) sideoats grama–inoculated–no fertilizer; (xi) sideoats grama–no inoculation–fertilizer; and (xii) sideoats grama–no inoculation–no fertilizer. Containers were obtained from Stuewe and Sons, Inc., Corvallis, OR, USA. Seeds of the grasses tested were obtained from La Campana Experimental Station, located
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75 km north of Chihuahua City in the State of Chihuahua, Mexico. For seed inoculation, the commercial product Azo-green was used, donated by Genesis and Turf and Forages, in Huntsville, UT, USA. Fertilizer was applied in a single application using the formula 30-0-0. The experimental design was a completely randomized block design with four replications. Each container was filled with 8 kg of dry soil sieved through a 2-mm screen. The containers had a bottom drainage hole covered with paper to prevent soil loss. The first experiment was planted on August 17. Eight pure live seeds were planted for each grass in each container at a depth of about 0.5–1 cm deep as evenly as possible. Prior to planting, seeds were inoculated with the Azo-green product with the amount recommended for expecting to deliver approximately 1 108 colony formed units g 1 of seed. After seeding, soils were watered to reach field capacity, and a second irrigation was done 9 days later to assure seed emergence. After emergence the containers were thinned leaving four homogenous seedlings per container. For the experiment in 1993, above-ground phytomass was handclipped on September 23, at 1 cm ground level and these data were not analysed. In November 23, forage production was evaluated through cutting at ground level in each container, and then roots were also carefully washed to estimate root phytomass. Dry Matter yield for both top and underground phytomass was determined after oven-drying the samples. Nitrogen percentage was determined by the Kjeldahl method, phosphorous was analysed by spectrometry, and calcium values were obtained by the titrimetry method for the samples obtained in the first experiment (Kalra, 1997). Crude protein was also measured (Sullivan and Carpenter, 1993). The second experiment was seeded in March 5, 1994 following the same method as the first experiment; therefore, containers were thinned 19 days after planting, a first cut was done after 35 days and the data were not analysed, the above-ground as well as underground phytomass were evaluated 60 days after the first cutting. An analysis of variance (Statgraphic) was conducted for above-ground as well as for root phytomass in each experiment. When the analysis of variance was significant, group contrasts were used to denote treatment differences, using 0.01 probability levels.
3. Results and discussion The soil was a sandy loam with a pH of 7.9, without salt problems (0.57 dS m 1 electrical conductivity), nor sodium problems (0.44 SAR). Nitrogen (NO3–N) was 5.5 meq l 1, phosphorous (NaHCO3 extract) was 7.9 meq l 1, potassium (1:5 extract soil–water) was 33 meq l 1; and iron was 5.3 meq l 1. In addition, the soil analysis gave levels of sodium of 0.65 meq l 1; calcium of 2.98 meq l 1, and magnesium of 1.27 meq l 1. Significant differences were detected in above-ground phytomass among species (Factor A) as well as for inoculation treatments (Factor B); however, surprisingly no significant differences were noted for fertilizer applications (Factor C) in both experiments (Table 1). In addition, for the experiment conducted in 1993, a strong
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Table 1 Above-ground phytomass of three grass species inoculated with A. brasilense First experiment (1993)
Second experiment (1994)
g
95% interval
g
95% interval
(A) Grass species Wilman lovegrass Weeping lovegrass Sideoats grama
5.6849a 2.2079b 1.2832c
5.1979; 6.1719 1.7200; 2.6940 0.7962; 1.7702
5.7262a 3.2193b 1.8987c
5.5772; 5.8752 3.0703; 3.3684 1.7497; 2.0477
(B) Inoculation Inoculated No inoculated
3.3744a 2.7424b
2.9767; 3.7720 2.3444; 3.1400
3.7054a 3.5241b
3.5837; 3.8271 3.4024; 3.6458
(C) Fertilizer Fertilizer No fertilizer
2.9604a 3.1563a
2.5628; 3.3580 2.7587; 3.5540
3.6762a 3.5533a
3.5545; 3.7979 3.4316; 3.6750
Means followed by the same letter are not significantly different at the 0.01 level of probability.
interaction was noted for both species–fertilization (AC), and inoculation– fertilization (BC), but no interaction was determined for species–inoculation (AB), nor for the triple interaction (ABC). For the experiment conducted in 1994, the different interactions were not significant. Of the three grasses evaluated, Wilman lovegrass had the best performance in terms of above-ground phytomass for both experiments with 5.68 g of dry matter and 5.72 g of dry matter, respectively; then weeping lovegrass with 2.20 g of dry matter and 3.21 g of dry matter, respectively. Sideoats grama had the lowest yields reaching 1.28 g of dry matter and 1.89 g of dry matter, respectively, for experiments conducted in 1993 and 1994 (Table 1). Inoculated treatments were different from non-inoculated treatments in both experiments. The experiment conducted 1993 resulted in 3.37 g of dry matter in inoculated treatments while non-inoculated treatments obtained 2.72 g of dry matter (Table 1). In the experiment conducted in 1994, the tendency among inoculated and non-inoculated treatments was similar to that of 1993 (Table 1). In both experiments above-ground phytomass was not affected for inorganic nitrogen application. The species–fertilization interaction was statistically different for the experiment conducted in 1993. The Wilman lovegrass’ above-ground phytomass increased slightly with fertilizer application. The above-ground phytomass for this species with nitrogen application was 5.94 g in comparison with 5.42 g without nitrogen application. A similar tendency was observed for sideoats grama, yielding 1.37 g with nitrogen application and 1.19 g without fertilization; however, the weeping lovegrass production was different. The above-ground phytomass production was 2.85 g without nitrogen application in comparison with 1.55 g with nitrogen application. The inoculum–fertilizer interaction was also significant. Inoculated treatments without fertilizer application performed better (3.87 g) than inoculated treatments with inorganic nitrogen applications (2.87 g). These results suggested that inoculation performed better than application of inorganic nitrogen fertilization.
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With respect to inoculation, these findings confirm that the bacteria are indeed more efficient when there is a lack of nitrogen in soil. These results agree with other findings that showed that inoculated treatments yielded more than control treatments (Zaady et al., 1994). Root phytomass was significantly different among species in both experiments (Table 2). Wilman lovegrass produced the most root phytomass reaching 3.20 g of dry matter in the first experiment while root phytomass in the second experiment was 3.15 g of dry matter. The weeping lovegrass performed second in terms of root phytomass obtaining 2.30 g of dry matter and 1.76 g of dry matter, respectively. Sideoats grama had the lowest root phytomass of the species that were tested in both experiments. This species reached 1.20 g of dry matter in Experiment 1, and 1.10 g of dry matter in Experiment 2. Significant differences were found for inoculated and no-inoculated treatments. The mean of inoculated treatments was 2.14 g of dry matter in the first experiment and 2.90 g of dry matter in the second experiment in comparison with 1.90 g of dry matter and 2.63 g of dry matter for non-inoculated treatments in Experiments 1 and 2, respectively. This result is extremely important for seeding rangeland areas where large, strong roots would have an increased chance of surviving. Surprisingly, the root phytomass was not affected by fertilizer applications. Fertilized treatments had 2.10 g of dry matter and 3.15 g of dry matter in Experiments 1 and 2 while nonfertilized treatments obtained 2.14 g dry matter and 3.01 g of dry matter for Experiments 1 and 2, respectively. Crude protein (CP) was lightly higher for sideoats grama than for Wilman lovegrass and weeping lovegrass. Mean CP for sideoats grama was 13% while Wilman lovegrass obtained 12% and weeping lovegrass reached 10.5%. Ca and P percentages did not differ among species. Ca percentage varied from 0.35% to 0.40%, while P ranged from 0.25% to 0.30%.
Table 2 Root phytomass of three grass species inoculated with A. brasilense
(A) Grass species Wilman lovegrass Weeping lovegrass Sideoats grama (B) Inoculation Inoculated No inoculated (C) Fertilizer Fertilizer No fertilizer
First experiment (1993)
Second experiment (1994)
g
95%interval
g
95%interval
3.2014a 2.3000b 1.2010c
2.6704; 3.7324 1.7690; 2.8310 0.6700; 1.7320
3.1510a 2.1315b 1.1000c
2.5996; 3.7024 1.5801; 2.6829 0.5486; 1.6514
2.1410a 1.9010b
1.7910; 2.4910 1.5510; 2.2510
2.9408a 2.6314b
2.7998; 3.0818 2.4904; 2.7724
2.1090a 2.1410a
1.7590; 2.4590 1.7910; 2.4910
3.1514a 3.0170a
3.0104; 3.2924 2.8760; 3.1580
Means followed by the same letter are not significantly different at the 0.01 level of probability.
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References Avivi, Y., Feldman, M., 1982. The response of wheat to bacteria of the genus Azospirillum. Israel Journal of Botany 31, 237–245. Barbieri, P., Zanelli, T., Galli, E., Zanetti, G., 1986. Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production. Federation of European Microbiological Societies’. Microbiology Letters 36, 87–90. Bashan, Y., Holguin, G., 1997. Azospirillum–plant relationships: environmental and physiological advances (1900–1996). Canadian Journal of Microbiology 43, 103–121. Bashan, Y., Ream, Y., Levanony, H., Sade, A., 1989. Nonspecific response in plant growth, yield, and root colonization of noncereal crop plants to inoculation with Azospirillum brasilense Cd. Canadian Journal of Botany 67, 1317–1324. Boddey, R.M., Baldani, V.L.D., Baldani, J.I., Dobereiner, J., 1986. Effect of inoculation of Azospirillum spp on nitrogen accumulation by field-grown wheat. Plant and Soil 95, 109–121. Bouton, J.H., Zuberer, D.A., 1979. Response of Panicum maximum to inoculation with Azospirillum brasilense. Plant and Soil 52, 585–590. ! para combatir la desertificaci!on en M!exico (PACD-MEXICO). Comision CONAZA, 1994. Plan de accion National de Las Zonas Aridas. Donahue, R.L., Miller, R.W., Shickluna, J.C., 1983. Soils—An Introduction to Soils and Plant Growth, 5th Edition. Prentice-Hall, Inc., Englewood Cliffs, NJ, 667pp. Eckert, B., Weber, O.B., Kirchhof, G., Halbritter, A., Stoffels, M., Harmann, A., 2001. Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C(4)-grass Miscanthus. International Journal of Systematic and Evolutionary Microbiology 51, 17–26. Estrada, B.W.J., 1987. Desertificacion en Mexico. Conferencia magistral dictada en el XX Congreso Nacional de la Ciencia del suelo Estrada. B.W.J. Zacatecas, M!exico, 20pp. Flach, K.W., Smith, G.O., 1969. The new system of soil classification applied to arid land soils. In: McGinnies, W.G., Goldman, B.J. (Eds.), Arid Land in Perspectives. The University of Arizona Press, Tucson, Arizona, 421pp. Givaudan, A., Bally, R., 1991. Similarities between large plasmida of Azospirillium lipoferum. Federation of European Microbiological Societies’ Microbiology Letters 78, 245–252. Hadas, R., Okon, Y., 1987. Effect of Azospirillum brasilense inoculation in root morphology and respiration in tomato seedlings. Biology and Fertility of Soils 5, 241–247. Haurat, J., Faure, D., Bally, R., Normand, P., 1994. Molecular relationship of an atypical Azospirillum strain 4T to other Azospirillum species. Research in Microbiology 145, 633–640. Hayes, D., 1991. Solar energy and desert development. In: Bishay, A. (Ed.), Desert Development SocioEconomic Aspects and Renewable Energy Applications. Gordon and Breach Publishing Group, London, 655pp. Hegazi, N.A., Khawas, H., Monib, M., 1981. Inoculation of wheat with Azospirillum under Egyptian conditions. In: Gibson, A.H., Newton, W.E. (Eds.), Current Perspectives in Nitrogen Fixation. Australian Academy of Science, Canberra, 493pp. Hegazi, N.A., Monib, M., Amer, H.A., Shokr, E.S., 1983. Response of maize plants to inoculation with Azospirilla and (or) straw amendment in Egypt. Canadian Journal of Microbiology 29, 888–894. Inbal, E., Feldman, M., 1982. The response of a hormonal mutant of common wheat to bacteria of the genus Azospirillum. Israel Journal of Botany 31, 257–263. Jenkinson, D.S., 2001. Nitrogen in a global perspective, with focus on temperate areas—state of the art and global perspectives. Plant and Soil 228, 3–15. Kadouri, D., Burdman, S., Jurkevitch, E., Okon, Y., 2002. Identification and isolation of genes involved in poly(b-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant. Applied and Environmental Microbiology 68, 2943–2949. Kalra, Y.P., 1997. Handbook of Reference Methods for Plant Analysis. CRC Press, Boca Raton, FL, 320pp. Khammas, K.M., Ageron, E., Grimont, P.A.D., Kaiser, P., 1989. Azospirillum irakense sp. nov., a nitrogen-fixing bacterium associated with rice roots and rhizosphere soil. Research in Microbiology 140, 679–693.
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Molina, J.D., 1983. Recursos agr!ıcolas de zonas a! ridas y semi!aridas de M!exico. Simposio. Centro de gen!etica, Colegio de Posgraduados, Chapingo, M!exico. Okon, Y., Itzigsohn, R., 1992. Poly-B-hydroxybutyrate metabolism in Azospirillum brasilense and the ecological role of PHB in the rhizosphere. Federation of European Microbiological Societies’. Microbiology Review 103, 131–140. Smith, R.L., Schank, S.C., Milam, J.R., Baltensperger, A.A., 1984. Response of sorghum and pennisetum species to the N2 fixing bacteria Azospirillum brasilense. Applied and Environmental Microbiology 47, 1331–1336. Sullivan, D.M., Carpenter, D.E. (Eds.), 1993. Methods of Analysis for Nutrition Labeling. Association of Official Analytical Chemist International, Gaithersburg, MD, 624pp. Tarrand, J.J., Krieg, N.R., Doberreiner, J., 1978. A taxonomic study of the Spirillum lipoferum group with descriptions of a new genus, Azospirillum gen, nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Canadian Journal of Microbiology 24, 967–980. Venkateswarlu, B., Rao, A.V., 1983. Response of pearl millet to inoculation with different strains of Azospirillum brasilense. Plant and Soil 74, 379–386. Zaady, E., Okon, Y., Perevolotsky, A., 1994. Growth response of Mediterranean herbaceous swards to inoculation with Azospirillum brasilense. Journal of Range Management 47, 12–15.
Above- and below-ground responses of Eragrostis and Bouteloua grass seedlings to the plant-growth-promoting bacterium Azospirillium brasilense He! ctor O. Rubio Ariasa, M. Karl Woodb,*, Carlos c ! Morales Nietoc, Gerardo Reyes Lopez , Lourdes de la Vegad a
Campo Experimental Madera, Instituto Nacional de Investigaciones Forestales, Agr!ıcolas y Pecuarias-SAGARPA, Ave. Homero No. 3744, Chihuahua, Mexico b Department of Animal and Range Science, New Mexico State University, Box 3-I, Las Cruces, NM 88003, USA c Campo Experimental la Campana, INIFAP-SAGARPA, Ave. Homero 3744, Chihuahua, Chihuahua C.P. 31100, Mexico d ! ! Facultad de Zootecnia de la Universidad Autonoma de Chihuahua, Kilometro 7.5 Carr. Chihuahua, Cuauht!emoc, Chihuahua, Mexico Received 27 January 2003; received in revised form 3 November 2003; accepted 14 January 2004
Abstract Two experiments were performed to determine the effects of inoculation with N2 fixing bacterium Azospirillum brasilense on forage production and root growth in seedlings of Wilman lovegrass, weeping lovegrass, and sideoats grama. Two sources of inorganic nitrogen (N) were tested: Urea, CO(NH2)2 (45%) and ammonium nitrate NH4NO3 (33.3%). Crude protein (CP), calcium (Ca) and phosphorous (P) concentrations were determined. In both experiments top dry matter (DM) production was significantly affected for grasses and inoculation but no differences were found for inorganic nitrogen applications. Maximum amount of top DM was obtained with the Wilman lovegrass with 5.70 g, weeping lovegrass reached 2.70 g, and sideoats grama only reached 1.58 g. Inoculated treatments reached 3.37 g while the no-inoculated treatments obtained 2.74 g. A similar trend was also noted for root phytomass. CP, Ca and P concentrations for the first experiment did not show any trend; therefore, they were not determined for the second experiment. Inoculation of grasses with
*Corresponding author. E-mail address: [email protected] (M.K. Wood). 0140-1963/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2004.01.008
ARTICLE IN PRESS 20
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A. brasilense may be a feasible practice for seeding some rangeland where N deficiency is a problem. r 2004 Elsevier Ltd. All rights reserved. Keywords: Nitrogen fixing bacteria; Azospirillium; Inorganic nitrogen; Root phytomass; Above-ground production
1. Introduction and literature Two decades ago, 40% of Mexico’s land was classified as arid or semi-arid (Molina, 1983) although more recent estimations consider that percentage to be as high as 52% (CONAZA, 1994). Across the world, arid and semi-arid land comprises about 40%, and it is believed that it is growing larger (Hayes, 1991). In addition, it is estimated that 6.7% of the Earth’s land has been turned into deserts; hence, it is expected that arid and semi-arid land will increase in Mexico as well as all over the world. Soil orders frequently found in arid and semi-arid environments include Aridisols, Entisols, Oxisols and Vertisols (Flach and Smith, 1969), which are low in organic matter, and as a consequence, low in nitrogen (N). It has been suggested for diverse ecological systems that the accumulation, degradation, and utilization of poly(bhydroxybutyrate) by several bacteria under stress is a mechanism that favors their establishment, proliferation, survival, and competition, especially in competitive environments where carbon and energy sources are limiting, such as those encountered in the soil and rhizosphere (Kadouri et al., 2002; Okon and Itzigsohn, 1992). In Mexico, a study showed that 70% of all soils have less than 1% organic matter (Estrada, 1987); therefore, the main problem in arid areas, without considering water supply is low levels of N. Although the atmosphere is 78% N, this element cannot be used by plants (Donahue et al., 1983) with the exception of two cases: N fixing by the symbiosis of the complex Leguminosae–Rhizobium that depends on formation of nodules in roots and for some N fixing bacteria that have the ability to fix N without nodulation. Recent information showed that world production of N fertilizer has overtaken the other sources of supply to plants (Jenkinson, 2001). Nitrogen fixing bacteria have been broadly studied because of their biological as well as their economic importance in areas such as medical use, degradation of pollutants, vitamin production, and purification of residual water (Bashan and Holguin, 1997). Few studies has been conducted on perennial grasses (Eckert et al., 2001) and even fewer by inoculating perennial plant species found in rangeland areas. The genera Azospirillum, Azotobacter, and Clostridiumn belong to the Azotobacteraceae family, which are bacteria that fix nitrogen without forming nodules in the plants’ roots. Azospirillum is aerobic and possesses a metabolism where the O2 or the NO3 are the electron acceptors (Haurat et al., 1994). Presently, there are five different species: Azospirillum brasilense, A. lipoforum, A. amazonense, A. halopraeferens, and A. irakense (Tarrand et al., 1978; Khammas et al., 1989;
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Givaudan and Bally, 1991). Bouton and Zuberer (1979) inoculated the bacteria A. brasilense to the grass Panicum maximum and noted an increase in biomass production. They also found that inoculation increased nitrogen content in the plant tissue. Other research conducted in Israel (Avivi and Feldman, 1982; Inbal and Feldman, 1982), Egypt (Hegazi et al., 1981; Hegazi et al., 1983), and Brazil (Boddey et al., 1986) obtained similar results. Moreover, some researchers have suggested that certain bacteria, such as Azospirillum, might provide higher yields in cereals (Smith et al., 1984) as well as in some non-cereal crop plants (Bashan et al., 1989; Zaady et al., 1994). The above-mentioned effects of bacteria inoculation can result in more root volume (Barbieri et al., 1986), an increase in root biomass (Hadas and Okon, 1987) and enlarged and healthier root systems (Venkateswarlu and Rao, 1983). The largest portion of Azospirillum literature consists of genetic studies of almost all aspects of the bacterium and its association with plants (Bashan and Holguin, 1997). To date little information is available on the effects of inoculating free living bacteria on non-cereal and perennial grass species’ yields (Bashan and Holguin, 1997); therefore, the objective of this research was to determine the effects of inoculating A. brasilense on the seeds of Wilman lovegrass (Eragrostis superba Peyn.), weeping lovegrass (E. curvula [Schrad.] Nees) and sideoats grama (Bouteloua curtipendula [Mich.] Torr.) with and without applying inorganic nitrogen fertilizer. These results are of practical importance to northern Mexico because these grass species are recommended for seeding rangelands.
2. Materials and methods Two experiments were conducted in the greenhouse of the Faculty of Zootechnic of the Autonomus University of Chihuahua, Mexico, during 1993 and 1994. The soil was collected in an undisturbed area and analysed in the Soil and Water Testing Lab at New Mexico State University in October 18, 1993. Experiments were conducted in 48 containers (19.5 cm diameter, 31.70 cm height) containing 8 kg of dry soil. In both experiments, the seeds of the grasses to be tested were inoculated with the bacteria A. brasilense. The first experiment evaluated application of urea CO(NH2)2 (45%), while in the second experiment the application of ammonium nitrate NH4NO3 (33.3%) as inorganic nitrogen was evaluated. A 3 2 2 factorial arrangement of treatments (three grass species, two levels of inoculation and two levels of inorganic nitrogen application) was employed. Hence, treatments were the followings: (i) Wilman lovegrass–inoculated–fertilizer; (ii) Wilman lovegrass–inoculated–no fertilizer; (iii) Wilman lovegrass–no inoculation–fertilizer; (iv) Wilman lovegrass– inoculation–no fertilizer; (v) weeping lovegrass–inoculated–fertilizer; (vi) weeping lovegrass–inoculated–no fertilizer; (vii) weeping lovegrass–no inoculation–fertilizer; (viii) weeping lovegrass–no inoculation–no fertilizer; (ix) sideoats grama–inoculated– fertilizer; (x) sideoats grama–inoculated–no fertilizer; (xi) sideoats grama–no inoculation–fertilizer; and (xii) sideoats grama–no inoculation–no fertilizer. Containers were obtained from Stuewe and Sons, Inc., Corvallis, OR, USA. Seeds of the grasses tested were obtained from La Campana Experimental Station, located
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75 km north of Chihuahua City in the State of Chihuahua, Mexico. For seed inoculation, the commercial product Azo-green was used, donated by Genesis and Turf and Forages, in Huntsville, UT, USA. Fertilizer was applied in a single application using the formula 30-0-0. The experimental design was a completely randomized block design with four replications. Each container was filled with 8 kg of dry soil sieved through a 2-mm screen. The containers had a bottom drainage hole covered with paper to prevent soil loss. The first experiment was planted on August 17. Eight pure live seeds were planted for each grass in each container at a depth of about 0.5–1 cm deep as evenly as possible. Prior to planting, seeds were inoculated with the Azo-green product with the amount recommended for expecting to deliver approximately 1 108 colony formed units g 1 of seed. After seeding, soils were watered to reach field capacity, and a second irrigation was done 9 days later to assure seed emergence. After emergence the containers were thinned leaving four homogenous seedlings per container. For the experiment in 1993, above-ground phytomass was handclipped on September 23, at 1 cm ground level and these data were not analysed. In November 23, forage production was evaluated through cutting at ground level in each container, and then roots were also carefully washed to estimate root phytomass. Dry Matter yield for both top and underground phytomass was determined after oven-drying the samples. Nitrogen percentage was determined by the Kjeldahl method, phosphorous was analysed by spectrometry, and calcium values were obtained by the titrimetry method for the samples obtained in the first experiment (Kalra, 1997). Crude protein was also measured (Sullivan and Carpenter, 1993). The second experiment was seeded in March 5, 1994 following the same method as the first experiment; therefore, containers were thinned 19 days after planting, a first cut was done after 35 days and the data were not analysed, the above-ground as well as underground phytomass were evaluated 60 days after the first cutting. An analysis of variance (Statgraphic) was conducted for above-ground as well as for root phytomass in each experiment. When the analysis of variance was significant, group contrasts were used to denote treatment differences, using 0.01 probability levels.
3. Results and discussion The soil was a sandy loam with a pH of 7.9, without salt problems (0.57 dS m 1 electrical conductivity), nor sodium problems (0.44 SAR). Nitrogen (NO3–N) was 5.5 meq l 1, phosphorous (NaHCO3 extract) was 7.9 meq l 1, potassium (1:5 extract soil–water) was 33 meq l 1; and iron was 5.3 meq l 1. In addition, the soil analysis gave levels of sodium of 0.65 meq l 1; calcium of 2.98 meq l 1, and magnesium of 1.27 meq l 1. Significant differences were detected in above-ground phytomass among species (Factor A) as well as for inoculation treatments (Factor B); however, surprisingly no significant differences were noted for fertilizer applications (Factor C) in both experiments (Table 1). In addition, for the experiment conducted in 1993, a strong
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Table 1 Above-ground phytomass of three grass species inoculated with A. brasilense First experiment (1993)
Second experiment (1994)
g
95% interval
g
95% interval
(A) Grass species Wilman lovegrass Weeping lovegrass Sideoats grama
5.6849a 2.2079b 1.2832c
5.1979; 6.1719 1.7200; 2.6940 0.7962; 1.7702
5.7262a 3.2193b 1.8987c
5.5772; 5.8752 3.0703; 3.3684 1.7497; 2.0477
(B) Inoculation Inoculated No inoculated
3.3744a 2.7424b
2.9767; 3.7720 2.3444; 3.1400
3.7054a 3.5241b
3.5837; 3.8271 3.4024; 3.6458
(C) Fertilizer Fertilizer No fertilizer
2.9604a 3.1563a
2.5628; 3.3580 2.7587; 3.5540
3.6762a 3.5533a
3.5545; 3.7979 3.4316; 3.6750
Means followed by the same letter are not significantly different at the 0.01 level of probability.
interaction was noted for both species–fertilization (AC), and inoculation– fertilization (BC), but no interaction was determined for species–inoculation (AB), nor for the triple interaction (ABC). For the experiment conducted in 1994, the different interactions were not significant. Of the three grasses evaluated, Wilman lovegrass had the best performance in terms of above-ground phytomass for both experiments with 5.68 g of dry matter and 5.72 g of dry matter, respectively; then weeping lovegrass with 2.20 g of dry matter and 3.21 g of dry matter, respectively. Sideoats grama had the lowest yields reaching 1.28 g of dry matter and 1.89 g of dry matter, respectively, for experiments conducted in 1993 and 1994 (Table 1). Inoculated treatments were different from non-inoculated treatments in both experiments. The experiment conducted 1993 resulted in 3.37 g of dry matter in inoculated treatments while non-inoculated treatments obtained 2.72 g of dry matter (Table 1). In the experiment conducted in 1994, the tendency among inoculated and non-inoculated treatments was similar to that of 1993 (Table 1). In both experiments above-ground phytomass was not affected for inorganic nitrogen application. The species–fertilization interaction was statistically different for the experiment conducted in 1993. The Wilman lovegrass’ above-ground phytomass increased slightly with fertilizer application. The above-ground phytomass for this species with nitrogen application was 5.94 g in comparison with 5.42 g without nitrogen application. A similar tendency was observed for sideoats grama, yielding 1.37 g with nitrogen application and 1.19 g without fertilization; however, the weeping lovegrass production was different. The above-ground phytomass production was 2.85 g without nitrogen application in comparison with 1.55 g with nitrogen application. The inoculum–fertilizer interaction was also significant. Inoculated treatments without fertilizer application performed better (3.87 g) than inoculated treatments with inorganic nitrogen applications (2.87 g). These results suggested that inoculation performed better than application of inorganic nitrogen fertilization.
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With respect to inoculation, these findings confirm that the bacteria are indeed more efficient when there is a lack of nitrogen in soil. These results agree with other findings that showed that inoculated treatments yielded more than control treatments (Zaady et al., 1994). Root phytomass was significantly different among species in both experiments (Table 2). Wilman lovegrass produced the most root phytomass reaching 3.20 g of dry matter in the first experiment while root phytomass in the second experiment was 3.15 g of dry matter. The weeping lovegrass performed second in terms of root phytomass obtaining 2.30 g of dry matter and 1.76 g of dry matter, respectively. Sideoats grama had the lowest root phytomass of the species that were tested in both experiments. This species reached 1.20 g of dry matter in Experiment 1, and 1.10 g of dry matter in Experiment 2. Significant differences were found for inoculated and no-inoculated treatments. The mean of inoculated treatments was 2.14 g of dry matter in the first experiment and 2.90 g of dry matter in the second experiment in comparison with 1.90 g of dry matter and 2.63 g of dry matter for non-inoculated treatments in Experiments 1 and 2, respectively. This result is extremely important for seeding rangeland areas where large, strong roots would have an increased chance of surviving. Surprisingly, the root phytomass was not affected by fertilizer applications. Fertilized treatments had 2.10 g of dry matter and 3.15 g of dry matter in Experiments 1 and 2 while nonfertilized treatments obtained 2.14 g dry matter and 3.01 g of dry matter for Experiments 1 and 2, respectively. Crude protein (CP) was lightly higher for sideoats grama than for Wilman lovegrass and weeping lovegrass. Mean CP for sideoats grama was 13% while Wilman lovegrass obtained 12% and weeping lovegrass reached 10.5%. Ca and P percentages did not differ among species. Ca percentage varied from 0.35% to 0.40%, while P ranged from 0.25% to 0.30%.
Table 2 Root phytomass of three grass species inoculated with A. brasilense
(A) Grass species Wilman lovegrass Weeping lovegrass Sideoats grama (B) Inoculation Inoculated No inoculated (C) Fertilizer Fertilizer No fertilizer
First experiment (1993)
Second experiment (1994)
g
95%interval
g
95%interval
3.2014a 2.3000b 1.2010c
2.6704; 3.7324 1.7690; 2.8310 0.6700; 1.7320
3.1510a 2.1315b 1.1000c
2.5996; 3.7024 1.5801; 2.6829 0.5486; 1.6514
2.1410a 1.9010b
1.7910; 2.4910 1.5510; 2.2510
2.9408a 2.6314b
2.7998; 3.0818 2.4904; 2.7724
2.1090a 2.1410a
1.7590; 2.4590 1.7910; 2.4910
3.1514a 3.0170a
3.0104; 3.2924 2.8760; 3.1580
Means followed by the same letter are not significantly different at the 0.01 level of probability.
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