ABSTRACT: In recent years the cultivation of cacao (Theobroma cacao L.) in Colombia has been growing up, resulting in the need to develop a sustainable production system. In this regard, ben eficial soil microorganisms are an alternative for improving plant productivity, but this requires knowledge of their ecology and functioning. This study had the objective of identify and quantify arbuscular mycorrhizal fungi (AMF) and fluorescent Pseudomonas sp. associated with the soil and rhizosphere of cacao plants in two agroecosystems, one of them was in a tropi cal dry forest (TDF) and the other in a tropical moist forest (TMF). In a second stage of the study, native strains of Glomus sp., Acaulospora sp., and fluorescent Pseudomonas sp. were se lected and multiplied in the lab. Subsequently, the effectiveness of these strains to promote cacao seedling growth was tested in a greenhouse experiment. The results indicate that there was a significant (P<0.05) greater mycorrhizal colonization and diversity associated to the roots of cacao growing in agroeco systems of the tropical moist forest. However, not significant differences were detected regarding the presence of fluorescent Pseudomonas sp. in the two agroecosystems. Otherwise, in the greenhouse experiments, the inoculation with the mycorrhizal fungus Glomus sp. was the only treatment that promoted the cacao seedling growth.
Key words: tropical dry foresttropical dry forest,tropical moist foresttropical moist forest,Glomus spGlomus sp.
RESUMEN: En los últimos años, el cultivo de cacao (Theobroma cacao L.) en Colombia viene creciendo, esto hace necesario desarrollar un sistema de producción sostenible. Es así, como los micro organismos benéficos del suelo son una excelente alternativa para mejorar la productividad de la planta, pero es importante conocer su funcionamiento y ecología. Este trabajo tuvo como objetivo identificar y cuantificar hongos micorrízicos arbus culares (HMA) y Pseudomonas sp. fluorescente associados al suelo y la rizosfera de plantas de cacao en dos agroecosistemas, bosque seco tropical (BST) y bosque húmedo tropical (BHT). En una segunda etapa del trabajo se seleccionaron y multiplicaron cepas nativas de Glomus sp., Acaulospora sp., y Pseudomonas sp. fluorescente en el laboratorio. Asi la efectividad de estas cepas para promover el crecimiento de plántulas de cacao bajo condiciones de invernadero fue evaluada. Los resultados indican que hubo una diferencia significativamente (P<0.5) mayor en la colonización micorrizal y diversidad asociada a raíces de cacao creciendo en el agroeosistema asociado al bosque húmedo tropical. Sin embargo, no se encontraron dife rencias significativas asociados a la presencia a Pseudomonas sp. fluorescente asociados a los dos agroecosistemas. Por otro parte en los experimentos en invernadero, la inoculación con el hongo micorrízico Glomus sp. fue el único tratamiento que promovió el crecimiento de plántulas de cacao.
Palabras claves: bosque húmedo tropical, bosque seco tropical, Glomus sp.
Agroecology
Presence of mycorrhizal fungi and a fluorescent Pseudomonas sp. in the rhizosphere of cacao in two agroecosystems and their effects on cacao seedling growth
Presencia de hongos micorrízales y una Pseudomonas sp. fluorescente en la rizosfera de cacao en agroecosistemas y sus efectos en el crecimiento de plántulas de cacao
Universidad Nacional de Colombia, Facultad de Agronomía
Received: 09 June 2016
Accepted: 30 June 2016
Cacao (Theobroma cacao L.) is a neotropical plant spe cies originated from the humid tropics of America, and thus presents great diversity in this zone (Enriquez, 2004; Motamayor et al. 2008). In Colombia, the cacao is cultivated in soils with different biological, physical, and chemical characteristics (Suarez et al., 2010). Most of the soils in the tropical areas present low nutrient availability, which results in low crop productivity (Brady and Weil, 2008; Meason et al., 2009; Osorio, 2011). This condition leads to add high doses of chemical, mineral, and organic fertil izers in order to improve yields, but the excessive use of these supplies can increase productions costs and generate negative environmental impacts (Brady and Weil, 2008; Xiao et al., 2008).
Arbuscular mycorrhizal fungi (AMF) are considered to be of great importance for adequate plant development, since they can improve water and nutrient uptake, particu larly phosphorus (P) (Osorio, 2011; Ramírez et al., 2013; Ramírez et al., 2014; Ramírez et al., 2015). This is relevant because in the tropics approximately 71% of plant species form arbuscular mycorrhizal association, 16% form other types of mycorrhizal associations, and only 13.4% are non-mycorrhizal (Coyne, 2000; Barea and Azcon, 2002). Among the first ones, it is well known the ability of cacao plants to form mycorrhizal associations and increase plant growth (Azizah-Chulan and Martin, 1992).
On the other hand, some bacteria from the Pseudomonas genus are known as plant growth promoters. They can colonize plant root systems and promote plant growth by (i) nutrient solubilization (e.g., P, Fe, among others), (ii) synthesizing hormonal regulators such as auxins and gibberellins (Gunes et al., 2015; Pii et al., 2015) and (iii) decrease phytopathogens populations in the soil and thus prevent plant diseases (Hallmann et al., 1997; Van Veen et al., 1997; Lugtenberg and Kamilova, 2009).
However, few researches exist about the presence of both types of these microorganisms in cacao plantations in contrasting environmental conditions, as those imposed by the TDF and TMF life zones, which certainly limits the application of these microorganisms at a commercial scale (Cuadros et al., 2011). For this reason, the present study had two objectives (i) identifying and quantifying AMF and strains of fluorescent Pseudomonas sp. in the rhizosphere of cacao plants in two agroecosystems of contrasting life zones: tropical dry forest (TDF) and tropical moist forest (TMF); and (ii) to evaluate the effectiveness of inoculation with these two groups of microorganisms on cacao plant growth in greenhouse.
Soil samples were collected from two sites: (i) Santa Fe de Antioquia at the Cotove Agriculture Experimental Station (altitude 540 m a.s.l., annual rainfall 900 mm, mean air temperature 27°C and relative humidity 75.5%) located at 6°32'55"N and 75°50'3"W, which corresponds the TDF ecological life zone according to Holdridge (1967); (ii) Ma ceo at the Theobroma farm (altitude 1,050 m a.s.l., annual rainfall 2,500 mm, mean air temperature 20°C, relative humidity 93%) located at 6°32'43"N and 74°47'27"W, which represent the TMF ecological life zone. Samples were transported to the Soil Laboratory of the Universidad Nacional de Colombia at Medellin for microbial processing. Greenhouse experiments were carried out in the same university (6°15'47"N and 74°34'40"W).
In each location four plots were selected and five trees of the IMC-67 (rootstock) and CCN-51 (scion) clones were randomized and chosen for sampling their rhizosphere. Surface soil (0-30 cm) and root fragments were taken 1 m away from the tree stem; from each plot 1 kg of soil and 50 g of roots were collected. Soil sampling was conducted at the Soil Fertility Laboratory at the same university; the results are shown in table 1 href="?v34n3a10"> .
Soil fertility parameters from each cacao agroecosystem.
Methods: texture by Bouyoucos; soil pH measured in water (2:1, V:V); soil organic matter (SOM) measured by Walkley and Black; Al extracted by 1 M KCl; Ca, Mg, and K extracted by 1 M ammonium acetate; P extracted by Bray II; soil solution P measured in 0.01 M CaCl2.
AMF spores were isolated using the wet sieving and de canting method described by Gerdemman and Nicholson (1963). Briefly, it consists of passing 20 g of soil sample through a set of sieves (425, 90, and 25 µm). The portion retained in each sieve was suspended with 50 mL of water and then centrifuged for two min at 2,000 rpm. The pre cipitate was suspended in 50 mL of a 50% sucrose solution and centrifuged again for five min at 4,000 rpm. The su pernatant obtained was passed through a filter paper for spore counting using a microscope (Nikon Eclipse E200). AMF identification was based in taxonomic keys (Clap et al., 1995; Heijden et al., 2004; Peterson et al., 2004). The Shannon diversity index was constructed using the PAST program, version 2.16 (Hammer et al., 2001).
Root mycorrhizal colonization was conducted after clear ing root fragments (1 cm length) with 10% KOH for 24 h (Phillips and Hayman, 1970) and then settled down in an alkaline solution (0.5% NH4OH and 0.5% H2O2) for 30 minutes (Brundrett and Abbott, 1995). Root fragments were washed with tap water and then acidified with 10% HCl for five min. After that, roots fragments were stained with 0.025% trypan, blue dissolved in lacto-glycerol (Kormanik et al., 1980) and the extension of roots colonized by the AMF was measured by the line-intercept method (Giovannetti and Mosse, 1980).
Rhizosphere samples were diluted with sterile water up to 10-4 serial dilution; 100 |iL of the 10-3 and 10-4 serial dilu tions were transferred onto King B (KB) culture medium in Petri dishes (King et al., 1954). The grouth medium was supplemented with ampicillin (50 mg L-1) and chlo-ramphenicol (12.5 mg L-1) (Simon and Ridge, 1974). Petri dishes were incubated for 48 h at 28°C. Positive colonies were considered those capable of producing fluorescent pigment under ultraviolet light (260 nm) (Ramírez, 2005).
Surface soil sample (0-30 cm) was collected from the Carimagua Experimental Station (USDA Soil Taxonomy Oxisol, Haplustox). It was analyzed in the Soil Fertility Laboratory at the same university and with the same methods. Soils results were: sand 480, silt 200, and clay 320 g kg-1 (Bouyoucos), pH 4.8 (water, 2:1), SOM 60 g kg-1, Al 0.9 cmolc kg-1 (1M KCl); Ca, Mg, and K 1.1, 0.4, and 0.2 (1 M ammonium acetate).
In order to avoid microbial interference, the soil was steril ized in an autoclave at 0.1 MPa and 121°C, for two cycles of one hour each. Lime was added to adjust soil pH at 5.6, based on the lime incubation method (Uchida and Hue 2000). Based on a P sorption isotherm (Fox and Kamprath, 1970), KH2PO4 was added to achieve a soil solution P con centration of 0.02 mgL-1, which is considered optimal for mycorrhizal activity.
At 50 d seeds of the IMC-67 clone germinated in peat until developing 5 leaves. Then two seedlings were transplanted into plastic pots containing 2 kg of the soil. One month later, one plant was removed. The plants were kept for other four months in greenhouse conditions and were frequently watered to maintain soil moisture content between 50-60% of the soil's maximum water retention capacity. Every week 50 mL of a P-free Hoagland solution was added.
For this experiment, we used two mycorrhizal inocula, one inoculum contained spores of the genus Glomus sp. and the other spores of the genus Acaulospora sp., it is also called Rhizoglomus (Sieverding et al., 2015) and Rhizofagus (Schufiler and Walker, 2010). These were selected since both genera were the most abundantly in the plots evaluated in the field. Both AMF were multiplied in corn roots until reaching a minimum concentration of 45 infective propa-gules per g of inoculum (Porter, 1979).
Several isolates of fluorescent Pseudomonas sp. were tested for their capability to produce indoleacetic acid using Salkowski indicator solution (Gordon and Weber, 1951). The solution was modified for use in bacterial culture supernatants (Patten and Glick, 2002). Additionally, the bacteria Ralstonia solanacearum was used as a control strain (C). This was conducted to select the isolate with the highest production of indole-acetic acid. The isolate selected (designed as P10) was multiplied in KB medium for 48 h at 28°C and suspended in sterile water; the bacterial suspension contained 1x106 colony forming units (CFU) per mL (Jena, 2012).
The treatments evaluated were: (i) inoculation with Acau lospora sp. (designated as "A") at a rate of 70 g of inoculum per 2 kg of soil and mixed throughout, (ii) inoculation with Glomus sp. (designated as "G") at a same rate; (iii) inoculation with fluorescent Pseudomonas sp. (P10) at a rate of 5 mL per plant; (iv) inoculation with Acaulospora sp. + fluorescent Pseudomonas sp. (A+P10) at the same rates described above; (v) inoculation with Glomus sp. + fluo rescent Pseudomonas sp. (G+P10); (vi) an not inoculated control (control) was included as a reference. All inocula were added at the transplanting time. Each treatment had five replicates.
The response variables were: plant dry weight after oven-dry a 60°C for 72 h, foliar P concentration using the non destructive method developed by Aziz and Habte (1987), and root mycorrhizal colonization as described above. Additionally, the fluorescent Pseudomonas sp. bacterium was recovered from the rhizosphere in order to confirm its presence.
In each experiment (sampling and greenhouse) a completely randomized design was used. Levene and Kolmogorov-Smirnov criteria were used to confirm data homoscedasticity and normality (P<0.05), respectively. The Ramirez, Osorno, and Osorio: Presence of mycorrhizal fungi and a fluorescent Pseudomonas sp. in the rhizosphere of cacao in two agroecosystems and their effects... data were subjected to analysis of variance and the Tukey test for mean separation both with a significance level P<0.05. Statistical analysis was performed with the soft ware R (R version 3.2.3 (2015-12-10) "Wooden Christmas-Tree" Copyright (C) 2015 The R Foundation for Statistical Computing Platform: i386-w64-mingw32/i386 (32-bit).
The two agroecosystems exhibited a significant difference in soil parameters; for instance, soil pH in Maceo was extremely acidic (4.7), whereas in Santa Fe the soil pH was neutral (6.7) (Tab. 2). Consistently, the soil solution P in Maceo was very low (0.011 mg L-1) and in Santa Fe was high (0.240 mg L-1). Despite of these contrasts, not significant difference was detected in the foliar P concentration of cacao plants. Similarly, there was not significant difference in the CFU of Pseudomonas spp.
The soil test results are consistent with the typical soil fertility parameters commonly found in these two life zones. For instance, Maceo soils are very acidic and poor in available P as a result of leaching out of bases (Ca, Mg, K, Na) produced by a high rainfall regime and is rich in Al as a result of high weathering rates, which generate oxides of Al and Fe that held P in unavailable forms for plant roots. By contrast, Santa Fe soil has a neutral soil reaction and is rich in available P, this associated with low weathering, low rainfall and the consequent dominance of 2:1 clays (e.g., montmorillonite, vermiculite) that retain high amounts of exchangeable bases (Zapata, 2002).
Mycorrhizal colonization was significant different between both agroecosystems, in Maceo was 12.1% and in Santa Fe was 3.7%. Accordingly, the number of AMF spores was significantly higher in Maceo (16.2) than in Santa Fe (12.8).
Both agroecosystems showed similar AMF genera, but their relative distribution was different (Fig. 1). Although, Glomus genus is more abundant than the other genera, in Maceo it represented almost half AMF population, while in Santa Fe it was three quarters of the AMF population. Acaulospora genus was the second in abundance in both agroecosystems (9-24%), followed by Gigaspora (8-11%) and by the other two genera (Scutellospora, Entrophospora). These differences were satisfactorily reflected in Shanon Index (Tab. 2), which was significantly higher in Maceo (1.82) than in Santa Fe (1.30).
FIGURE 1
Relative distribution of AMF genera in two agroecosystems of cacao.
Variables evaluated in the two agroecosystems.
Means followed by different letters indicate significant differences according to the Tukey test (P0.05).1 MC: mycorrhizal colonization; 2 Spores per g of soil.
The bacterium that was used as control R. solanacearum did not produce indole-acetic acid. In contrast, all Pseudomo nas isolates did produce it, but the concentration produce was variable among them (Fig. 2). The isolate P10 was the most active in this regard (79.9 µg mL-1) followed by P9, P3 and P8 (48.9-59.3 µg mL-1), and then by P1, P2, and P3 (29.5-38.9 µg mL-1). The less effective isolate were P4, P5 and P6 (20.2-25.0 µg mL-1).
FIGURE 2
Indoleacetic acid produced by different isolates of fluorescent Pseudomonas sp. isolated from cacao rhizospheres; C = control ino culated with R. solanacearum. T1-T10 = number of isolated strains. Means with different letters indicate significant differences according to the Tukey test (P<0.05).
The findings of this study coincide with those of Prieto et al. (2012) who found AMF species of Glomus, Scutellospora, Acaulospora and Gigaspora in cacao agroforestry systems placed in a TDF in Ecuador. However, these authors did not report Entrophospora genus. In a study by Cuenca and Meneses (1996) in Venezuela the genus most associated with cacao was Glomus, being G. etunicatum particularly prominent. According to Sieverding (1991), Glomus is one of the most competitive and effective AMF genus in with different botanical groups and different soil conditions. In this study, clearly Glomus is dominant in both agroecosys tems and in the case of any inoculation with AMF, species of Glomus should be considered. This was confirmed with the greenhouse results, in which Glomus was consistently effective to promote plant P uptake and growth (Zandavalli et al., 2004; Osorio 2011; Ramírez et al., 2013; Ramírez et al., 2014; Ramírez et al., 2015).
The MC values reported here coincide with those from Bolívar et al. (2009), who found MC between 2.8 and 13.3% under natural conditions. The former value might be considered low, since Cuenca et al. (1991) reported MC values up to 69%. Meanwhile, in agroforestry systems in a TMF life zone in Ecuador, Prieto et al. (2012) reported that MC did not surpass 3.5%, while values were as low as 0.9%. Perhaps, the high levels of soil available P found in Santa Fe agroecosytems may restrict the extent of mycor rhizal colonization (Peters and Habte, 2001), while the low levels of this in Maceo may promote a high mycorrhizal colonization. Cuenca et al. (1991) reported that in soils with low nutrient availability, cacao plants are highly dependent on AMF. This is because under these conditions, the plant requires this positive symbiosis for the uptake of this nu trient (Bonfante and Genre, 2010; Barea and Pozo, 2013).
Cuadros et al. (2011) reported that in field conditions MC depends largely on soil P content. López etal. (2007), Toro et al. (2008), and Azizah-Chulan and Martin (1992) reported that the recommended commercial dose of P fertilizers for cacao had negative effect on native AMF populations. They recommended adjusting the P fertilization management in order to promote the AMF symbiosis. On the other hand, Tena (2002) affirms that high levels of Ca and Mg (as those found in Santa Fe) may affect the AMF spores density and thus the symbiotic relationship with the host plant.
Despite the presence of fluorescent Pseudomonas sp., we did not find any promoting effects on cacao plants in the greenhouse experiment. It is not clear if in the two agro-ecosystems they had a role on plant nutrition or growth, it has been reported that they are biocontrol agents and plant growth promoters (Hallmann et al., 1997; Van Veen et al., 1997; Lugtenberg and Kamilova, 2009). Notice that in cacao plants at Maceo the foliar P was as good as in Santa Fe, in despite of that the former one was grown in a P deficient soil. It may be explained by the high levels of MC, the role of this bacterium in those systems should be investigated.
Uninoculated plants (control) showed a plant dry weigh of 150.9 g, which was significantly increased (+36%) only by Glomus inoculation. Other inoculation did not increase significantly the plant growth of cacao plant, included the co-inoculation G+P10 (Fig. 3A).
FIGURE 3
Variables evaluated in greenhouse conditions in cacao seed lings inoculated with an AMF. A: Acaulospora sp. G: Glomus sp. in combination with fluorescent Pseudomonas sp. (P10). Means with different letters indicate significant differences according to the Tukey test (P<0.05).
Control plants exhibited a foliar P concentration of 0.43%, this was significantly higher when the mycorrhizal fungi were inoculated (Fig. 3B). Inoculated plants with Acau-lospora had foliar P concentration of 0.67% and those inoculated with Glomus had a concentration even higher 0.92%, which represented relative increases of 56 and 114%, respectively. The inoculation with Pseudomonas sp. (P10) was ineffective to promote foliar P (0.51%). Moreover, the co-inoculation with P10 was not effective to increase the foliar P beyond that obtained with the AMF inoculation only. In this way, the foliar P concentration of plants treated with A+P10 was 0.73% (similar to A-inoculation) and with G+P10 was 0.86% (similar to G-inoculation).
Uninoculated plants and those inoculated with P10 did not have mycorrhizal colonization (Fig. 3C). In contrast, the plants inoculated with Acaulospora (without and with P10) showed a mycorrhizal colonization that ranged be tween 15.8-13.4%, respectively, which did not differ to each other. Otherwise, the inoculation with Glomus had values of mycorrhizal colonization significantly higher than the other with values that fluctuated between 32.5-37.9 %, without and with P10, respectively.
Inoculation of the IMC-67 clone with Glomus sp. resulted in higher MC values than those reported by Ballesteros et al. (2004) (ca. 20%) with native Glomus and Acaulospora. Similarly, Cuadros et al. (2011) reported MC values of 84% with both AMF genera. These results indicate that the MC of cacao roots by AMF inoculated is variable. From our perspective, this variable (MC) may be secondary, and it is necessary to focus on the effectiveness of AMF inocula tion on plant nutrient uptake and growth or even better on crop yield.
In this study it is clear that, at least for cacao seedlings, the use of AMF inoculation promote growth, as reported by Azizah-Chulan and Martin (1992). However, this effect seems to be species-dependent. Thus, species of Glomus must be included in a commercial inoculum intended to promote seedling growth and P uptake. The benefits ob tained with Glomus inoculation are quite relevant since the P foliar was twice higher with this than in control plants. On the other hand, the effect of Pseudomonas sp. P10 was null alone or in combination with AMF.
In both cacao agroecosystems, AMF and Pseudomonas were detected. The same AMF genera was found but in different relative distribution. The AMF genus Glomus was predominant in both agroecosystems. On the other hand, the number of CFU of Pseudomonas spp. was similar in both agroecosystems. In the greenhouse, cacao seedlings had higher levels of foliar P when inoculated with AMF (Acaulospora and Glomus) than uninoculated plants, but the effect was higher with Glomus species. Plant growth was only significantly promoted by Glomus inoculation and not by Acaulospora. The inoculation with the isolate P10 of Pseudomonas did not promote plant growth and P uptake in mycorrhiza-free or mycorhizal plants.
3Corresponding author: jgramireg@unal.edu.co
Soil fertility parameters from each cacao agroecosystem.
Methods: texture by Bouyoucos; soil pH measured in water (2:1, V:V); soil organic matter (SOM) measured by Walkley and Black; Al extracted by 1 M KCl; Ca, Mg, and K extracted by 1 M ammonium acetate; P extracted by Bray II; soil solution P measured in 0.01 M CaCl2.
FIGURE 1
Relative distribution of AMF genera in two agroecosystems of cacao.
Variables evaluated in the two agroecosystems.
Means followed by different letters indicate significant differences according to the Tukey test (P0.05).1 MC: mycorrhizal colonization; 2 Spores per g of soil.
FIGURE 2
Indoleacetic acid produced by different isolates of fluorescent Pseudomonas sp. isolated from cacao rhizospheres; C = control ino culated with R. solanacearum. T1-T10 = number of isolated strains. Means with different letters indicate significant differences according to the Tukey test (P<0.05).
FIGURE 3
Variables evaluated in greenhouse conditions in cacao seed lings inoculated with an AMF. A: Acaulospora sp. G: Glomus sp. in combination with fluorescent Pseudomonas sp. (P10). Means with different letters indicate significant differences according to the Tukey test (P<0.05).
