SUELOS ECUATORIALES 39 (1): 100-106

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ARTCULO DE INVESTIGACIN CIENTIFCA Y TECNOLOGICA MYCORRHIZAL DEPENDENCY OF COFFEE SEEDLING AT DIFFERENT LEVELS OF SOIL SOLUTION PHOSPHORUS
Sandra P. Jaramillo & Nelson W. Osorio Departamento de Geociencias, Universidad Nacional de Colombia, Medelln, Colombia Calle 59A No. 63-20 Of. 14-238, Medelln, Colombia. Tel. 574-4309310, Telefax: 574-4309311
*Corresponding author: nwosorio@unal.edu.co

ABSTRACT
A greenhouse experiment was carried out to determine the mycorrhizal dependency (MD) of coffee (Coffea arabiga) seedlings at different levels of soil solution P. With this purpose coffee seedlings cv. Caturra and Colombia were grown for 144 days in a soil either inoculated or not with Glomus fistulosum at three concentrations of soil solution P (0.005, 0.02 and 0.2 mg L-1). The results indicated that both coffee cultivars were highly dependent on the mycorrhizal association. The response of coffee seedlings to mycorrhizal inoculation varied with the level of soil solution P. At 0.005 mg L-1, coffee seedlings exhibited a poor growth and there was not significant increase on P uptake and shoot dry mass with the mycorhrizal inoculation. At 0.02 mg L-1 mycorrhizalfree seedlings did not grow well but the inoculation significantly increased the plant P uptake and dry weight of both cultivars. At 0.2 mg L-1, mycorrhizal-free seedlings exhibited a good growth, but the mycorrhizal inoculation significantly decreased plant P uptake and shoot dry weight of both varieties. These results suggest that coffee seedlings would benefit considerably from AMF inoculation if the level of soil solution P is adequate (0.02 mg L-1). Key words: phosphorous, arbuscular mycorrhiza, Coffea arabiga, Caturra, Colombia, Glomus fistulosum

DEPENDENCIA MICORRIZAL DE PLANTULAS DE CAF A DIFERENTES NIVELES DE FSFOR EN SOLUCIN

RESUMEN Un experimento de invernadero se realiz para determinar la dependencia micorrizal de plntulas de caf (Coffea arabiga) a diferentes niveles de fsforo (P) en la solucin del suelo. Para este prposito plntulas de caf de la variedades Caturra y Colombia crecieron durante 144 das en un suelo inoculado o no con el hongo micorrizal Glomus fistulosum a tres concentraciones de P en la solucin del suelo (0.005, 0.02 y 0.2 mg L-1). Los resultados indican que ambas variedades fueron altamente dependientes de la asociacin micorrizal. La respuesta de la planta a la inoculacin micorrizal vari con el nivel de P en la solucin del suelo. A 0.005 mg L-1, las plntulas de caf crecieron pobremente y no hubo incremento significativo en la absorcin de P ni en el crecimiento de las plntulas de caf con la inoculacin micorrizal. A 0.02 mg L-1 las plntulas de caf nomicorrizadas no exhibieron un buen crecimiento pero con la inoculacin micorrizal se increment significativamente la absorcin de P y la masa seca de las plntulas de ambas variedades de caf, siendo el efecto mayor en la variedad caturra. A 0.2 mg L-1, las plntulas de caf no-micorrizadas mostraron un buen crecimiento, pero con la inoculacin micorrizal significativamente disminuy la absorcin de P y la masa seca de ambas variedades. Los resultados claramente indican que las plntulas de caf pueden derivar beneficios de la inoculacin micorrizal si el nivel de P en la solucin del suelo es adecuado (0.02 mg L-1). Palabras claves: fsforo, arbuscular micorriza, Coffea arabiga, Caturra, Colombia, Glomus fistulosum

INTRODUCTION The growth and development of coffee (Coffea arabiga) seedlings can be impaired by the low soil availability of phosphorus (P) in the Andean mountains of Colombia (Avila et al. 2007). Many of these soils are acid, aluminium rich, and some are formed from volcanic ashes and characterized by the dominance of allophane in the clay fraction (Malagon et al. 1991).
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These soils exhibit high capacity to sorb P onto their surface (Shoji et al. 1993) and, consequently, it is necessary to apply large amounts of P fertilizers (Osorio 2008), which increases production costs and environmental concerns (Havlin et al. 1999; Holford 1997). The use of arbuscular mycorrhizal fungi (AMF) can enhance plant P uptake and growth of several plant species (Allen, 1996; Brundrett et al., 1996; Harrier

S. P. Jaramillo & N.W. Osorio

2001) including coffee (Arango et al. 1992; Orozco 1988; Osorio et al. 2002; Siqueira et al. 1993). The use of AMF in coffee crop is very promising because this is an environmental-friendly practice that can reduce P fertilizers application and improve plant growth. However, little is known about the mycorrhizal dependency (MD) of coffee cultivars that are commonly grow in Colombia. Our hypothesis is that given the coarse root system of coffee, this plant species must exhibit certain degree of dependency on the mycorrhizal association and this can be affected by the level of soil Pi bioavailability. The purpose of this study was to determine the MD of coffee cv. Caturra and Colombia and the interaction between Glomus fistulosum inoculation and soil solution Pi concentration on plant growth and P uptake. MATERIALS AND METHODS The study was conducted in the greenhouse of the Soil Microbiology Laboratory (615N, 7535W, 1495 m altitude) of the National University of Colombia at Medelln. A sub-superficial (30-50 cm) soil sample from a Bt horizon of an Ultisol, collected from the El Volador mountain at Medellin, was air-dried, sieved through a 4mm sieve, mixed with quartz (soil:quartz ratio of 2:1), and autoclaved at 120C and 0.1 MPa for one hour. Based on a chemical soil analysis the following compounds were applied (per kg): 2 g calcium carbonate, 436 mg of ammonium sulfate, 1550 mg of calcium sulfate, 980 mg of magnesium sulfate, 5 mg of FeEDTA, 5 mg of Cu-EDTA, % mg Zn-EDTA, and 5 g borax. According to the approach of Habte and Manjunath (1991) for MD determination, a soil P sorption isotherm (Fox and Kamprath 1970) was conducted to determine the P requirement to achieve three levels of soil solution P (0.005, 0.02, and 0.2 mg L1 ). As a result of that, KH2PO4 was added at 0, 950 and 2800 mg kg-1, respectively, and mixed uniformly. To balance the level of potassium added, 1533 and 1066 mg of potassium per kg of substrate were applied at the lowest and medium level of added P. Plastic bags (17x23 cm) were filled with 1.8 kg (dry basis) of the amended and sterile mixture of soil and quartz. The substrate was either inoculated (M+) or not inoculated (M-) with 50 g of a crude mycorrhizal inoculum, which was mixed through out. The inoculum contained 11 infective propagules of Glomus fistulosum per g, which was determined by the most probable number technique (Porter 1979). The inoculum was multiplied in a mixture of soil: quartz (1:1, m/m) with sorghum and kudzu as host plants. Uninoculated soil (M) received 50 g of sterilized substrate and 20 cm3 of washing from the crude inoculum after remotion of

mycorrhizal fungi structures by filtration with filter paper Whatman No. 1. Seedlings of coffee were obtained from seeds and grown in a sterile substrate for 60 days. Then, the seedlings of both cultivars were transplanted into the Pamended and inoculated substrate. A thin layer of fine quartz was applied on the surface of each pot to prevent contamination by mycorrhizal spores from surroundings or other treatments. Seedlings were growing under natural light for 144 days after transplanting. The substrate was watered with distilled water to maintain it at 50-60% of the maximum water holding capacity. To prevent nutritional deficiencies in plants, we supplied 50 cm3 of P-free Hoagland solution to each bag once a week. The bags were moved on a weekly basis to randomize variation of environmental conditions. A complete randomized experimental design was employed, the treatments were arranged in a factorial combination 2x3x2: two coffee cultivars, three levels of soil solution P, and two levels of mycorrhizal inoculation (M+, M-). Each treatment had four replications. Foliar P content were monitored 68, 106, 135, and 144 days after transplanting. The foliar P content was monitored using the non-destructive sampling method developed by Aziz and Habte (1987). For this purpose, a leaf-disk (6 mm of diameter) was collected from the youngest mature leaf. The leaf-disk samples were ashed in a muffle furnace at 500C for three hours, then the ash was dissolved in distilled water. The P concentration was measured by the blue-molibdate method (Murphy and Riley, 1962). At harvest, the shoot dry weight (SDW) was determined after oven-dry the samples at 60C for 96 h. Fine roots were cleaned with water, cleared by immersion in KOH (10 % m/v in water) for 24 h (Phillips and Hayman, 1970). Root samples were stained with fucshin acid (0.15%) (Kormanik et al. 1980). To determine root colonization by the AMF we used the method of grid-line intersection (Giovannetti and Mosse, 1980). The MD was evaluated as proposed by Plenchette et al. (1983) as the difference between shoot dry mass of inoculated and uninoculated plants, expressed as a percentage of shoot dry mass of inoculated plants. The mean value of MD obtained at the soil solution P of 0.02 mg L-1 was compared with the categories proposed by Habte and Manjunath (1991). The data were statistically analyzed with Statgraphics Plus version 4.0 employing analysis of variance and LSD test to separate the effect of inoculation at each level of soil P (P-value 0.05). RESULTS Only those plants grown in the inoculated substrate exhibited mycorrizal colonization. This parameter was not affected by the level of soil solution P and ranged

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between 30 and 40% regardless the treatments. The foliar P content of coffee seedlings was significantly (P<0.05) affected by the treatments, however the response to AMF inoculation was controlled by the level of soil solution P. For instance, at 0.005 mg L-1 there was an increase in the foliar P content of both cultivars with the inoculation at any sampling date (Figure 1). By contrast, at 0.02 mg L-1 the mycorrhizal inoculation significantly increased the P content in the leaf disk of both cultivars Caturra and Colombia in all sampling dates (Figure 1). The magnitud of these increase ranged from 1.9 to 3.6 for cv. Caturra, and 2.1 to 3.0 for cv. Colombia. On the other hand, at 0.2 mg L-1 the foliar P content in the leaf-disks of both cultivars significantly decreased with the AMF inoculation, which was detected in all sampling dates (Figure 1). The shoot dry weight (SDW) exhibited a similar pattern to the foliar P content. At the level of soil solution P of 0.005 mg L-1 the AMF inoculation did not increase the SDW of both cultivars (Figure 2). At 0.02 mg L-1 the inoculation significantly increased the SDW of both cv. Caturra and Colombia, which were 3.5 and 2.0-times higher than their respective uninoculated controls (Figure 2). By contrast, at 0.2 mg L-1 the AMF inoculation significantly decreased the SDW of both cultivars (Figure 2). The cv. Caturra that grow in the inoculated soil exhibited 17% of the SDW of the inoculated control, while the reduction was down to 21% for the cv. Colombia. The value of the mycorrhizal dependency (MD) of both cultivars was significantly affected by the level of soil solution P (Table 1). At the lowest level of soil available P, the MD was 23.4 and 10.7 % for Caturra and Colombia, respectively. At 0.02 mg L-1 the value of the MD reached a peak of 71 and 50.1%, respectively. At 0.2 mg L-1, given the reduction of SDW of both cultivars with the AMF inoculation the values of MD were negtive (Table 1). Both coffee cultivars can be classified as highly dependent on the mycorrhizal association.
Table 1. Mycorrhizal dependency (MD) of coffee plants cv. Caturra and Colombia at different concentration of soil solution P. Soil solution P MD (%) of cv. MD (%) of cv. Caturra Colombia (mg L-1) 0.005 0.02 0.2 23.4 71.0 -463.6 10.7 50.1 -362.4

DISCUSION The results clearly showed that coffee seedlings of both cultivars exhibited a high degree of MD, but this

was significantly affected by the soil solution P concentration. The soil solution P concentration controls the response of plant to mycorrhizal inoculation (Habte and Manjunath 1991; Johnson et al. 1997). For instance, at very low concentration of soil P the inoculation did not have effect on plant growth and P uptake despite of the fungal colonization in the roots as found by other authors (Gonzalez and Osorio, 2008; Plenchete and Morel, 1996). It is possible that the P concentration of 0.005 mg L-1 is below the detection level of P transporters in the cell membrane of the fungi (Habte and Manjunath 1987). In contrast, at 0.02 mg L-1 the response of coffee plants to the inoculation was outstanding, which suggest that this concentration is adequate for the fungus but is still low to the mycorrhizafree roots as reported by several authors (Diez et al. 2006; Gonzalez and Osorio 2008; Habte and Manjunath 1987). Our results agreed to those found by Habte and Biitenbender (1999), who reported that coffe cv. Typica exhibted a very high MD (MD >75%) on the fungus G. aggregatum. On the other hand, the P concentration in soil solution of 0.2 mg L-1 was very favorable to the unaided roots given the plant growth observed. However, at this high concentration of soil P the inoculation had a negative effect (Figure 1 and 2). This reduction in plant development with the AMF inoculation has been observed with other plant species (Habte and Soedarjo 1996; Habte and Byappanahalli, 1994; Miyasaka et al., 1993; Plenchette and Morel, 1996; Siqueira and SagginJnior 2001). It seems that the excess of P in solution decreases the bioavailability of other nutrient (e.g., Fe, Mn, Cu, and Zn) (Havlin et al., 1999), which may affect the activity of both symbionts (Marschner 1997). Also, Fitter (1991) affirms that the response to the mycorrhizal association is controlled by the exchange of carbonaceous compounds by P between plant and fungus. The plant gives up to 20 to 30% of the C fixed in the photosynthesis to the fungus that is used to satisfy its nutrtional requirement (Jakobsen and Rosendahl 1990). As the fungal hypha grows it can capture the escarce soluble P, which is translocated into the root cells (Allen 1996).Under this condition the plant does not require the fungus in the roots and the fungal nutritional needs constitutes a C-drainage for the host plant without an apparent benefit to the plant. Smith et al. (2003) and Burleigh et al. (2002) reported that once the mycorrhizal fungus is established in the root tissue, the root cells inhibit their capacity to absorb P relying on the fungus to capture it. Nevertheless, at high levels of soil solution P the plant inhibt the activity and extent of the fungus (Harrison 1999; Harrison and Van Buuren 1995; Johnson et al. 1997; Liu et al. 1998; Maldonado-Mendoza et al..

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3 cv. Caturra
Foliar P content (g per leaf disk)

3 MFoliar P content (g per leaf disk)

(0.005 mg L-1) 2

M+

cv. Colombia (0.005 mg L-1) 2

0 50 70 90 110 130 150

0 50 70 90 110 130 150

Time (days)

Time (days)

3 cv. Caturra (0.02 mg L-1) 2

3 cv. Colombia (0.02 mg L-1)

0 50 70 90 110 130 150

0 50 70 90 110 130 150

Time (days)

Time (days)

3 cv. Caturra (0.2 mg L-1) 2

3 cv. Colombia (0.2 mg L -1) 2

0 50 70 90 110 130 150

50

70

90

110

130

150

Time (days)

Time (days)

Figure 1. Foliar P content in leaf-disk of coffee cv. Caturra and Colombia grown in a soil inoculated (M+) or inoculated (M-) with G. fistulosum at three levels of soil solution P in different time after transplanting. The LSD values (P <0.05) for the days 68, 106, 135, and 144 were 0.17, 0.23, 0.37, and 0.37, respectively.

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5 cv. Caturra Shoot dry weight (g per plant) 4

M+

5 cv. Colombia 4

LSD 0.05

M-

0 0.005 0.02 Soil solution P (mg L -1) 0.2

0 0.005 0.02 Soil solution P (mg L )

-1

0.2

Figure 2. Shoot dry weight of coffee plants cv. Caturra and Colombia grown in a soil inoculated (M+) or inoculated (M-) with G. fistulosum at three levels of soil solution P. LSD (P <0.005) = 0.47.

2001; Murphy et al. 1997; Peng et al. 1993; Rausch et al. 2001; Rosewarne et al. 1999), which creates an unbalance between C supply and nutrient uptake. In summary, the results of this study clearly show that the AMF inoculation can increase plant growth and P uptake at moderate concentration of soil available. It also shows that the AMF can reduce the need for P fertilizers. Note that the amount of P added to obtain 0.02 mg L-1 was 33% of that requiered to achieve 0.2 mg L-1. This can represent a reduction in cost production, especially with the notorious increase of P fertilizers in recent years (Hylton, 2008). Also, it is clear that the cultivar Caturra can obtain more benefits that the cv Colombia, such differences have been reported by other authors with several plant species (Bryla and Koide 1990, 1998; Khalil et al. 1994; Monzn and Azcn 1996; Smith et al. 1990, 1999). ACKNOWLEDGEMENTS. This work was finnacially
supported by the Direction of Research (DIME) and the Laboratory of Soil Fertility of the Universidad Nacional de Colombia at Medelln.

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Recibido: Enero 15 de 2009 Aceptado: Mayo 20 de 2009

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