versão On-line ISSN 0717-6643
Gayana Bot. v.66 n.2 Concepción 2009
Gayana Bot. 66(2): 206-217, 2009
EFFECTS OF INORGANIC FERTILIZERS AND RHIZOBIAL INOCULATION ON GROWTH, NODULATION AND TANNIN CONTENT OF ACACIELLA ANGUSTISSIMA (MILL.) BRITTON & ROSE
EFECTO DE LOS FERTILIZANTES INORGÁNICOS Y LA INOCULACIÓN RIZOBIAL SOBRE CRECIMIENTO, NODULACIÓN Y CONTENIDO DE TANINOS EN ACACIELLA ANGUSTISSIMA (MILL.) BRITTON & ROSE
Víctor M. Ruiz-Valdiviezo1, Teresa R. Ayora-Talavera1, Federico A. Gutiérrez-Miceli1, Luc Dendooven2 & Reiner Rincón-Rosales1
1 Departamento de Biotecnología Vegetal, Instituto Tecnológico de Tuxtla Gutiérrez, Tuxtla Gutiérrez, Carretera Panamericana km 1080, C.P. 29050, Chiapas, México;
2 Laboratory of Soil Ecology, Department of Biotechnology and Bioengineering, Cinvestav, Av. I.P.N. 2508, C.P. 07360 México DF, México. firstname.lastname@example.org
The effects of different inorganic fertilizers and rhizobial inoculation on shoot height, total shoot and root dry weight, nodule number, total shoot nitrogen, nitrogenase activity and tannic acid content of Acaciella angustissima (Mill.) Britton & Rose were investigated in the laboratory. Seedlings were grown in a climate chamber in glass tubes containing sterilized mixture of vermiculite and peat moss, and treated with combinations of nitrogen (N) at 45 mg plant1, phosphorus (P) at 30 mg plant1, potassium (K) at 20 mg plant1 and inoculated with the bacterium Sinorhizobium mexicanum. The combined applications of N, P or K to uninoculated plants increased shoot height and dry weight as compared to the unamended plantlets. The tannin content in uninoculated plants was highest when amended with P+K. Treatment had a significant effect on plant growth, nodulation and tannin content varied. The plants treated with P + K + S. mexicanum had significantly longer shoot height, total shoot and root dry weight, nodule number, total shoot nitrogen, nitrogenase activity and tannic acid content in comparison with unamended plants. It was found that N reduced number of nodules, tannin content and nitrogenase activity of A. angustisisma. As such, farmers should refrain from applying N fertilizer, but could apply P and K to maximize tannin production in A. angustissima.
Keywords: Acetylene reduction, growth chamber, N P K fertilizer, Sinorhizobium mexicanum.
Se investigaron en el laboratorio los efectos de diferentes fertilizantes inorgánicos y la inoculación rizobial sobre la altura de planta, peso seco total de planta, peso seco de raíz, el número de nodulos, el nitrógeno total de la planta, la actividad de la nitrogenasa y el contenido de ácido tánico de Acaciella angustissima (Mili.) Britton & Rose. Las plántulas fueron crecidas en una cámara climática en tubos de vidrio conteniendo una mezcla de vermiculita y turba, y tratadas con las combinaciones de nitrógeno (N) en 45 mg planta"1, fósforo (P) en 30 mg planta"1, potasio (K) en 20 mg planta"1 e inoculado con la bacteria Sinorhizobium mexicanum. La combinación de las aplicaciones de N, P o K en plantas sin inocular incrementa la altura y el peso seco de planta en comparación con las plantas sin tratamiento. El contenido de taninos en plantas no inoculadas fue el más alto cuando suplementamos con P+K. El tratamiento tuvo un efecto significativo sobre el crecimiento de planta, modulación y el contenido de tanino variado. Las plantas tratadas con P + K + S. mexicanum tuvieron significativamente mayor altura de planta, peso seco total de planta, peso seco de raíz, el número de nodulos, nitrógeno total de planta, actividad de la nitrogenasa y contenido de taninos en comparación con las plantas sin suplementar. Fue encontrado que el N reduce el número de nodulos, el contenido de taninos y la actividad de la nitrogenasa de A. angustisisma. Como tal, los agricultores deberían abstenerse de aplicar el fertilizante de N, pero podrían aplicar P y K para maximizar la producción de tanino en A. angustissima.
Palabras clave: Reducción de acetileno, cámara de crecimiento, fertilizante N P K, Sinorhizobium mexicanum.
Acaciella angustissima (Mill.) Britton & Rose (Fabaceae) is a shrub that was classified as a Mimosa species and later as an Acacia (Rico-Arce & Rodríguez 1998). Now, it has been reclassified as Acaciella together with other American acacias (Rico-Arce & Bachean 2006). This species has a broad geographical distribution ranging from the Southern United States to Costa Rica, and in Mexico it can be found along the Pacific coast (Dzowela 1994). In Chiapas, A. angustissima shrubs are often used in agroforestry systems as they grow fast and have a high capacity to fix N2 because of their symbiotic relationship between roots and nitrogen fixing bacteria (Lloret et al. 2007; Rincón-Rosales et al. 2008). Interestingly, these shrubs are the preferred hosts of Llaveia mexicanorum (Williams & Mac Vean 1995), a native homeoptera scale insect, which is used by indigenous people of Chiapas and Mesoamerica to produce a fat for traditional lacquer wood hand crafts (Grillasca 2007). A. angustissima can grow under different climatic conditions in various ecosystems and in soils with different amounts of organic material and large differences in nutrient availability (Rincón-Rosales & Gutiérrez-Miceli 2008).
Inorganic N fertilizer can have an effect, being it positive or negative, on plant nodulation, N2 fixation and seedling growth of leguminous plants (Huda et al. 2007). Phosphorus has been shown to increase plant growth and stimulate nodulation in legumes (Rasanen & Lindstrom 2003). Potassium stimulates transport of molecules through the membrane, enzyme activity and cell growth (Ashley et al. 2006). Nitrogen is involved in cell division and biosynthesis of molecules in plant growth (Buchanan et al. 2000). High concentration of inorganic N in the soil normally inhibits symbiotic nitrogen fixation (Hungría & Vargas 2000), but not always (Davidson & Robson 1986) and changes the synthesis of phenolic compounds in legumes (Barahona et al. 1997). A. angustissima accumulates phenolitic compounds in its bark, i.e. tannins, and they are used as a defense against fungi and bacteria (O Donovan & Brooker 2001). Plants growing in nutrient-poor soil often contain high concentrations of tannins (Northup et al. 1995, Kraus et al. 2003, Kraus et al. 2004). In woody species, the concentration of foliar tannin commonly range from 15 to 25% dry weight (Booker & Maier 2001, Osier & Lindroth 2001), but amounts as high as 40% have been reported (Kuiters 1990, Matthews et al. 1997). As such, tannin could be extracted for industrial purposes. Tannins are traditionally used to tan hide. A. angustissima could be cultivated in marginalized land providing the farmers with an additional income while restoring soil fertility and preventing soil erosion. Fertilizing A angustissima might thus affect tannin content in its bark. As part of a project to reforestate and restore soil fertility in large parts of central Chiapas, the effect of N, P, K and Sinorhizobium mexicanum (Lloret et al. 2007) on growth, tannin and N content, nodulation and a nitrogenase activity of A. angustissima was studied in the laboratory.
MATERIALS AND METHODS
Seed collection, pretreatment and germination
Mature pods were collected from A. angustissima shrubs growing in the Sumidero Canyon National Park in Chiapas, Mexico (16° 48' N; 93° 04' W). The site is 900 to 1600 m above sea level on a gently sloping hill westerly orientated. The pods were sun-dried three days and the seeds extracted. Healthy seeds of uniform weight were scarified with H2S04 for 10 min and surface sterilized with 1 % (v/v) hypochlorite for 10 min (Rincón-Rosales et al. 2003). Treated seeds were germinated on 0.8 % agar-water plates at 28 °C in the dark for 48 h (Rincón-Rosales et al. 2008).
The S. mexicanum strain ITTG R7T was provided by the ITTG bacterial culture collection (Tecnológico de Tuxtla Gutierrez, Chiapas, México). This strain was isolated from A. angustissima root nodules in Chiapas and is a fast-growing rhizobia (Lloret et al. 2007). The strain was grown on yeast extract manitol agar (YEM) and peptone yeast (PY) media at 28 °C and preserved at 4 °C until used (Toledo et al. 2003).
Plant material and growth conditions
A. angustissima seeds were scarified with H2S04 for 15 min and the surface sterilized with 1% (v/ v) sodium hypochlorite for 10 min. Treated seeds were germinated on 0.8% agar-water plates and incubated in the dark at 25°C for 48 h to induce etiolation (Rincón-Rosales et al. 2003). The seedlings with a root of approximately 2 cm were placed in glass tubes (0 2 cm and length 20 cm), i.e. two seeds per tube, containing a mixture of sterilized vermiculite-peat moss (1:1) and moistened with a NPK-free Fahraeus solution (Fahraeus 1957). The tubes were closed first with a rubber-foam stopper and then with aluminium foil.
Treatments and experimental design
Sixteen different treatments were applied and four plants were used per treatment combining application of phosphorus (P), nitrogen (N), potassium (K) and inoculation with S. mexicanum (B) (Table I). Nitrogen was applied at 45 mg (NH4)2N03-N per tube, P at 30 mg NaH2P04-P and K at 45 mg K2S04-K at transplanting. The plants arranged completely at random were grown in a climate chamber at 28 °C with a 14 h light/ 10 h dark photoperiod (Rasanen & Lindstrom 2003). Lighting was provided by fluorescent lamps with 50 |o.mol m2 s ' light intensity. After 5 days, half of the plantlet roots were inoculated with 1 ml medium containing 10651. mexicanum ITTG R7T cells. Each fifteen days, all plants were irrigated with sterile 5 ml NPK-free Fahraeus solution.
After 90 days of cultivation, plants were taken from the glass tubes, the substrate was washed from the roots and shoot height, total dry weight and root dry weight was determined. Roots and shoots were oven-dried at 60°C for 48 h until a constant weight was obtained. The dry roots and shoots were hammer milled and an 80 mg sub-sample of the shoot was analyzed for total N by microKjeldahl (Bremmer 1996).
The effective nodules, i.e. red or brown coloured, were separated from the roots, cleaned, counted and weighed (Vincent 1970). Nitrogenase activity in the nodules (N2 fixation) was measured by the acetylene reduction assay (ARA) (Hardy 1968). The nodules were placed in a 25 ml vial, 600 jj.1 air was removed and replaced with acetylene (C2H2) and incubated at 30°C. After 2 h, the headspace of the vial was sampled and analyzed for ethylene production using a Varian model 3300 GC fitted with a flame ionization detector (FID) fitted with a porapak N column (300 x 0.1 cm). N2 fixation was expressed as micromoles of ethylene produced per nodule per h.
A 30 mg sub-sample of each root and shoot was analyzed for tannin content using the tungsten-molybdenum-phosphorus method (Miranda 2000).
Significant difference among plant characteristics as a result of the different treatments were determined by analysis of variance (ANOVA) and based on the least significant difference using the General Linear Model procedure PROC GLM (SAS Institute 1989). This procedure can be used for an analysis of variance (ANOVA) for unbalanced data, i.e. when some data are missing. Correlation coefficients were calculated with PROC CORR (SAS Institute 1989).
The relationships between the different plant characteristics, i. e. total plant N and tannin content, shoot height, root and above ground plant dry weight, number of nodules and nitrogenase activity, for plants not inoculated were analyzed by principal component analysis (PCA) using the orthogonal/ varimax rotation to achieve either small or large component loading and an eigenvalue of 1 as the lower limit. For those plants inoculated with S. mexicanum the number of nodules and nitrogenase activity were additionally included in the PCA analysis. Variables were auto-scaled prior to PCA (Sena et al. 2002). The number of components was determined by the Eigenvalue-one criterion (Kaiser 1960). Moreover, a scree test (Catell 1966) was performed to corroborate primer results, only principal components with Eigenvalues >1 and/or that explained >10% of the total variance were retained. A VARIMAX rotation was performed to enhance interpretability of the uncorrelated components (Flury & Riedwyl 1988). A PCA often reveals previously unsuspected associations among variables and thereby allows interpretation that would not be possible otherwise (Johnson & Wichern 1998). The matrix of 16 columns (treatments) and 7 (not inoculated) or 9 (inoculated with S. mexicanum) lines (variables) was used for principal component analysis. All analyses were performed using the SAS statistical package (SAS Institute 1989). All data presented were the mean of four plants cultivated (n=4).
Shoot height varied between 13.3 cm when only N and 21.1 cm when N+P+K was added (Table I). P had a positive highly significant effect on shoot height (P<0.0001) (Table II). Plant weight was the lowest when only P was added to the plants and the highest when inoculated with S. mexicanum and amended with P+K (Table I). P, K, S. mexicanum + N, S. mexicanum + P and N+K had a significant positive effect on plant weight (Table II). Root weight and total plant N content were lowest in the untreated plants and highest when inoculated with S. mexicanum and amended with P+K (Table I). P and the interactions between S. mexicanum and N and N+P+K had a significant effect on root weight (Table II). P and the interactions between P and K and S. mexicanum and N+P+K had a significant effect on total plant N (Table II). Tannin content of the plants was lowest when only P was added to the plants and highest when inoculated with S. mexicanum and amended with P+K (Table I ). All factors (P<0.0001) and interactions between them (P<0.05) had a significant effect on the plant tannin content, except for the interactions between S. mexicanum and P, and S. mexicanum and N+P (Table II). Seven nodules were found when plants were inoculated with S. mexicanum and amended with P+K, but only one when amended with N, N+P or N+K (Table I ). All factors (PO.0001) and interactions between them (P<0.05) had a significant effect on the nodule number, except for the N+P+K combination and the interaction between S. mexicanum and N+P+K (Table II). The lowest N2 fixing capacity was found when plants were inoculated with S. mexicanum and amended with N, and the highest when amended with P+K (Table I ). All factors (P<0.0001) and interactions between them (P<0.05) had a significant effect on the N2 fixing capacity of the plants, except for the interactions between S. mexicanum and N, S. mexicanum and K, S. mexicanum and N + P, S. mexicanum and N+K and S. mexicanum and N+P+K (Table II).
Tabla I. Efecto de los tratamientos sobre la altura de A. angustissima, el peso seco de planta y el peso seco raíz, el N total de planta y el contenido de taninos y el número de nodulos.
Table II. P-value for the effect of S. mexicanum, nitrogen, phosphorus, potassium and the interactions among them on height of A. angustissima, plant and root dry weight, total plant N and tannin content and number of nodules.
Tabla II. Valores de P para el efecto de Sinorhizobium mexicanum, nitrógeno, fósforo, potasio y las interacciones entre ellos sobre la altura de A. angustissima, el peso seco de planta y el peso seco raíz, el N total de planta y el contenido de taninos y el número de nodulos.
Principal component analysis
Loading for parameters obtained after VARIMAX rotation are given in Table III. A first PC (PCI), only considering the plants not inoculated with S. mexicanum, explained 37% of variation, a second 24% and a third 21%. PCI had a positive loading from root and plant weight, PC2 from shoot height and total plant N content and PC3 from tannin and total N content. On the scatter plot with PCI and PC2, the treatments are visually distinct (Fig. 1a). The unamended plants or those amended only with N, P or K were the lightest, i.e. a negative PCI value, while combination of the aforementioned nutrients resulted in heavier plants. The tallest plants and those with the highest N content, i.e. highest PC2, were found when plants were amended with the three nutrients. Plants with the largest tannin content, i.e. a high positive PC3, were obtained when fertilized with P+K (Fig. 1b).
Figure 1. Principal component analysis (PCA) performed on characteristics, i.e. shoot height, shoot and root weight, total N and tannin content of uninoculated A. angustissima. With A) depicting factor 1 and factor 2, while B) draws factor 1 and factor 3.
Figura 1. Análisis de componentes principales (PCA) realizado sobre la altura de planta, peso de planta, peso de raíz, N total y el contenido de tanino de A. angustissima no inoculada. Con A) representado el factor 1 y factor 2, mientras B) muestra el factor 1 y factor 3.
A first PC, only considering the plants inoculated with S. mexicanum, explained 54% of variation, a second 19% and a third 14% (Table III). PCI had a positive loading from N and tannin content, nitrogenase activity and number of nodules, PC2 from plant and root weight, and PC3 from root and plant weight and shoot height. On the scatter plot with PCI and PC2, the treatments are visually distinct (Fig. 2a). Fertilizing the plants with N reduced the number of nodules, nitrogenase activity and tannin content, compared to plants not amended with N. Adding P or K increased number of nodules, nitrogenase activity and tannin content, and combining both nutrients further increased these characteristics. Shoot height and weight was larger when all nutrients or N+P or P+K were added to the inoculated plants, i.e. positive PC2, compared to the other treatments. The plants with highest biomass were found when P+K was added to the inoculated plants (Fig. 2b).
Figure 2. Principal component analysis (PCA) performed on characteristics, i.e. shoot height, shoot and root weight, total N and tannin content, number of nodules and N2 fixing capacity, of A. angustissima inoculated with Sinorhizobium mexicanum after a varimax rotation. With A) depicting factor 1 and factor 2, while B) draws factor 1 and factor 3.
Figura 2. Análisis de componentes principales (PCA) realizado sobre la altura de planta, peso de planta, peso de raíz, N total, el contenido de tanino, número de nodulos y la capacidad de fijación de N de A. angustissima inoculada con Sinorhizobium mexicanum después de una rotación varimax. Con A) representado el factor 1 y factor 2, mientras B) muestra el factor 1 y factor 3.
A first PC, considering all treatments, explained 54% of variation, a second 19% and a third 15% (Table III). PCI had positive loading from root and plant weight, PC2 from shoot height and total plant N content and PC3 from plant tannin content. The unamended plants were the lightest, i.e. a negative PCI value, inoculating plants with S. mexicanum and adding P+K as nutrients resulted in the heaviest plants (Fig. 3a). The tallest plants and those with the highest N content, i.e. highest PC2, were found when plants were amended with the three nutrients. Plants with the largest tannin content, i.e. a high positive PC3, were obtained when inoculated and fertilized with P+K while generally N fertilizer or the absence of S. mexicanum reduced it (Fig. 3b).
Tabla III. Carga rotacional sobre los componentes principales.
Figure 3. Principal component analysis (PCA) performed on characteristics, i.e. shoot height, shoot and root weight, total N and tannin content, number of nodules and N2 fixing capacity of A. angustissima inoculated or not with Sinorhizobium mexicanum after a varimax rotation. With A) depicting factor 1 and factor 2, while B) draws factor 1 and factor 3.
Figura 3. Análisis de componentes principales (PCA) realizado sobre la altura de planta, peso de planta, peso de raíz, N total, el contenido de tanino, número de nodulos y la capacidad de fijación de N de A. angustissima inoculada o no con Sinorhizobium mexicanum después de una rotación varimax. Con A) representado el factor 1 y factor 2, mientras B) muestra el factor 1 y factor 3.
Nitrogen is one of the most critical nutrients for normal plant development. Leguminous plants have overcome the limitation of nitrogen by forming symbiotic relationship with N2 fixing bacteria (Rasanen & Lindstrom 2003). When no symbiotic relationship can be formed, such as in^. angustissima plants cultivated in sterile peat moss, N becomes the most limiting nutrient. In the peat moss used, there was a lack of P, but its positive effect on the development of A. angustissima was smallest. The combination of these nutrients stimulated morphological growth, biomass production, biological fixation of nitrogen and tannins content of A. angustissima seedlings.
Fertilizing legumes with N is known to inhibit nodule formation, but not always (Fujikake et al. 2003). Low concentration of inorganic N, i.e. ammonium or nitrate, have been shown to actually promote nodulation, but a larger concentration nearly always suppresses it (Davidson & Robson 1986). Legumes use soil N if available, because formation of nodules requires additional energy and nutrient substances from the plant (Turk et al. 1993). Soil nitrogen concentrations (trough roots) will regulate formation of nodules (Rhoades & Coleman 1999). Inorganic N added to A. angustissima cultivated in peat moss reduced nodule formation and nitrogenase activity.
Lack of inorganic N fertilizer increased tannin concentrations in A. angustissima. Plant tannin contents are affected by stress conditions (Kraus et al. 2004). A number of studies have examined changes in plant chemistry in response to variations in nutrients, light, moisture, ozone and C02 (Penuelas & Estiarte 1998, Hattenschwiler et al. 2003, Kraus et al. 2003). Most investigations found that phenolic and tannin concentrations increased when nutrient availability decreased and/or C availability increased. Plants growing in nutrient limited conditions often contain high concentrations of foliar tannins and other phenolics (Northup et al. 1998). This suggests that there may be a feedback wherein lack of nutrients increases secondary compound production leading to lower litter quality, which further contributes to poor soil conditions (Van Breemen & Finzi 1998). Plant secondary compounds, such as polyphenols and tannins, affect nutrient cycling in soil by inhibiting organic matter degradation, mineralization rate s and N availability (Kraus et al. 2003). Additionally, site characteristics have also been shown to affect plant phenolic concentrations (Covelo & Gallardo 2001). In the study reported here, the largest amounts of tannins were obtained when plants were inoculated and fertilized only with P+K. This indicated that not only inorganic N status in soil but also inoculation of A. angustissima with S. mexicanum might increase its plant tannin content.
P is essential for biosynthesis of nucleic acids, phospholipids, coenzymes and ATP all affecting photosynthesis, protein formation and N2 fixation (Buchanan et al. 2000). As such, plant development increases when P is added to plants. Additionally, P stimulates nodulation of legumes (Hellsten & Huss-Danell 2000), but how is still largely unknown.
Inoculation of A. angustisisma seedlings with S. mexicanum increased plant yield as much as did the addition of N, when seedlings were fertilized with P and K. It is difficult to speculate what might have caused this, but a lack of N might have been the reason.
It was found that N reduced number of nodules, tannin content and nitrogenase activity of A. angustisisma. As such, farmers should refrain from applying N fertilizer, but could apply P and K to maximize tannin production in A. angustissima.
We thank the plant biotechnology laboratory from the Technological Institute of Tuxtla Gutierrez (Chiapas, Mexico) for providing facilities for this investigation and the Centra de Ciencias Genómicas-UNAM (Cuernavaca, Morelos) for the determination of nitrogenase activity.
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