versión On-line ISSN 0718-9516
J. Soil Sci. Plant Nutr. vol.12 no.2 Temuco 2012
Journal of Soil Science and Plant Nutrition, 2012, 12 (2), 287-298
Effects of the co-inoculation with saprobe and mycorrhizal fungi on Vaccinium corymbosum growth and some soil enzymatic activities
C. Arriagada1*, D. Manquel1, P. Cornejo2, J. Soto1, I. Sampedro3, J. Ocampo3
1Laboratorio de Biorremediación, Departamento de Ciencias Forestales. Center of Amelioration and Sustainability of Volcanic Soils, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile.Corresponding author: firstname.lastname@example.org
2Departamento de Ciencias Químicas y Recursos Naturales. Center of Amelioration and Sustainability of Volcanic Soils, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera. Casilla 54-D. Temuco-Chile.
3Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, España.
In this study we evaluated the initial plant growth and some biochemical changes produced in rizhosphere soil of blueberry (Vaccinium corymbosum L.) cv. 'Elliot' plantlets obtained from in vitro culture and inoculated with six arbuscular mycorrhizal (AM) fungi strains (Gigaspora rosea; Glomus claroideum; G. deserticola; G. viscosum; G. intraradices and G. constrictum) and five saprobe fungi (Coriolopsis rigida; Phanerochaete chrysosporium; Trametes versicolor; Trichoderma harzianum and Penicillium chrysogenum). Our results showed a significant "like" AM root colonization (25-46%), highlighting the treatment co-inoculated with G. rosea + P chrysosporium or C. rigida; G. intraradices + C. rigida; G. viscosum + P chrysosporium, but this root colonization was morphologically atypical, and dominated by interstitial hyphae, without develop of other typical AM structures. Furthermore, significant increase in the shoot and root growth of the plantlets inoculated with G. viscosum + P chrysosporium, and G. intraradices + C. rigida were observed. On the other hand, G. claroideum + T. versicolor showed the high increase in the dehydrogenase enzyme activity, while inoculation with G. claroideum alone showed the greatest β-glucosidase activity. These results suggest that the interaction of AM symbionts together with saprobe fungi could be a biological alternative to promote the growth of micro-propagated blueberry plantlets in their early growth stages.
Keywords: Arbuscular mycorrhizae, biochemical properties, blueberries, plant symbionts, saprophytic fungi.
Vaccinium corymbosum L. (Ericaceae) is a shrub native from North American, which currently is extensive cropped in Chile. The surface under plantation has grown constantly since the introduction of the species into the country, reaching 12.000 ha. at present. In natural conditions this species grows in acidic soils, usually poor deficient and with high organic matter content. In this conditions, spontaneously forms mu-tualist symbiotic associations with certain soil fungi belonging mainly to the phylum Ascomycota, called "ericoid mycorrhizae" (Scagel et al., 2005; Smith and Read, 2008). Ericoid mycorrhizae would be specific to species of the families Ericaceae and Epacridaceae, due to their co-evolution with the native host plants growing in limiting edaphic conditions, allowing the plant to complete its life cycle (Brundrett, 2002). However, some fossil evidence indicates that originally the Ericales were able to establish arbuscular mycorrhizae (AM) (Brundrett, 2002), which have been reported in some species of this order, including several Vaccinium species (Koske et al., 1990).
Although these reports are scarce, and there is little in-depth studies in this sense (Brundrett, 2002), some authors suggest that the absence of ericoid mycorrhizal fungi inoculum may allows Ericales plants to associate with AM-forming fungi (Koske et al., 1990). Arbuscular mycorrhiza symbiosis is the most extended mutualistic association in nature, and unlike the ericoid mycorrhizae, it is not host-specific, being possible its formation between members of the phylum Glomeromycota and most of terrestrial plant species (van der Heijden, 2004; Singh, 2007).
In several micro-propagated plantlets, the application of mycorrhizal inoculants has proved to be effective improving their establishment and growth at transplantation, its resistance to hydric stress, its acquisition of mineral nutrients, and increasing its tolerance to pathogens (Scagel et al., 2005; Binet et al. 2007). Furthermore, the lapse for micro-propagation during their acclimatization period can also be reduced in mycorrhizal plants (Salamanca et al., 1992).
On the other hand, the saprobe fungi are important and common components of the rhizosphere. These fungi obtain a greater nutritional benefit when living roots release organic and inorganic compounds with sloughed cells (Dix and Webster, 1995). Additionally they produce plant growth-stimulating substances such as hormones and vitamins (Shetty et al., 1994). The saprobe fungi release energy and nutrients present in complex substrates in forms which can be assimilated by the plants by means the production of extracellular enzymes, and also are capable of degrading potentially phytotoxic substances (Arriagada et al., 2009a). In addition, some of them are able to produce substances that promote the growth of other microorganisms in the rhizosphere, including AM fungi (Fracchia et al., 2000).
For these reasons, we suggest that co-inoculation with AM and saprobe fungi in the acclimatization stage of micro-propagated blueberry plantlets will have a beneficial influence on their growth, therefore requiring a shorter-time for their establishment in the further field conditions. Thus, the aim of the present study was to evaluate the initial susceptibility of blueberry plantlets to colonization by AM fungi, as well as the effect exercised by saprobe fungi in improving this symbiosis and promoting the growth of blueberry plantlets in the acclimatization phase.
2. Material and methods
2.2 Plant species
After in-vitro culture (ten months) Vaccinium corymbosum cv. 'Elliot' uniform seedlings were planted in 1 L pots filled with a 1:4 mixture of sterilized sand and soil type Andisol, Serie Freire (pH 5.84; 10% organic matter; 13.7 mg kg-1 P-Olsen). The soil was sterilized at 100°C for 1 h for three consecutive days and distributed in 1000 mL pots. Plants were grown in a greenhouse with supplementary light provided by Sylvania incandescent and cool-white lamps (400 Em-2 s-1; 400-700 nm) with a 16/8 h day/night cycle at 25/19 °C and 50% relative humidity.
2.3 Arbuscular Mycorrhizal fungi
The AM fungi used in the experiment were Gigaspo-ra rosea Nicolson and Schenck; Glomus claroideum (Schenck and Smith) Walker and Vestberg; Glomus deserticola (Trappe, Bloss and Menge); Glomus viscosum T.H. Nicolson, Glomus intraradices Schenck and Smith and Glomus constrictum Trappe, obtained from the culture collection at the Laboratorio de Biorremediación at Universidad de La Frontera, Temuco, Chile. The AM inoculum was a root-and-soil inoculum consisting of rhizosphere soil containing spores and colonized root fragments of Medicago sativa L. in amounts of 10 g per pot, which were previously analyzed, showing a high potential to produce significant levels of root colonization. Plants uninoculated with AM fungi were given a filtrate (suspension of 50 g of fresh field soil in 1 l of distilled water for 10 min and filtered through a Whatman no. 1 paper) of the soil inoculum containing the common soil micro-flora (principally soil bacteria), but free of AM fungal propagules.
2.4 Saprobe fungi
The saprobe fungi Coriolopsis rigida (Berk. Et Mont.) Murrill, Phanerochaete chrysosporium Burds, Tram-etes versicolor (L.:Fr.) Pillat, Trichoderma harzianum Rifai and Penicillium chrysogenum Thorn., were obtained from the fungal collection at the Laboratorio de Biorremediación at Universidad de La Frontera, Temuco, Chile. The strains were stored on Malt Extract Agar (MEA) plates at 4 °C and periodically sub-cultured. To prepare the fungal inoculum, barley seeds were inoculated with a 1 cm2 disk of MEA withdrawn from 14 day-old fungal cultures grown at 28 °C. Ten barley seeds colonized by the mycelium of the fungi were added to the soil in each pot.
2.5 Experimental design
Four treatments were used: (1) Uninoculated plants (controls), (2) Soil inoculated with each saprobe fungi, (3) Soil inoculated with each AM fungi, and (4) Soil inoculated with each saprobe combined with each AM fungus. Plants were inoculated at the time of transplanting (after 4 weeks of growth). The saprobe fungi were inoculated at the same time as AM fungi. Three replicate per treatment were used.
2.6 Harvests and analyses
Plants were harvested after 14 weeks, dried in a air-forced oven (70°C, 48 h) and shoots and roots bio-mass were determined. After the harvest, fresh samples of roots were taken from the entire root system at random. The samples were cleared and stained using trypan blue in lactic acid (Phillips and Hayman, 1970) and the percentage of root colonized by mycorrhizal fungi was calculated by the gridline intersect method (Giovannetti and Mosse, 1980). The presence or absence of AM colonization at each intercept was used to determine the percentage of colonization and the colonized root length.
2.7 Biochemical determinations
To evaluate the biochemical changes in the rhizosphere, the enzymatic β-glucosidase and dehydrogenase activities were determined. Rhizosphere soil samples were collected after harvest at direct proximity of plant roots. The β-glucosidase was determined by measuring p-nitrophenol (PNP) released from p-nitrophenyl- β-D-
2.8 Statistical analysis
The percentage values were arcsine transformed for statistical analyses. Statistical procedures were carried out with the SPSS software, version 11.0 (SPSS Inc., 1989-2001). The data were analyzed by means of an analysis of variance (ANOVA) and the means were compared using orthogonal contrast test. Statistical significance was determined at P < 0.05.
3. Results and discussion
3.1 Root colonization
Results showed variations in intraradical AM colonization in V. corymbosum roots among the different AM fungal strains used, measured principally as appressoria, intracellular hyphal coils, arbuscules as intracellular arbusculate coils like branches and intraradical hyphae (Smith and Read, 2008) (Figure 1). In all the AM fungal strains studied, morphologically the fungus seems to begin the symbiotic growth, but in no case were clearly distinguished the typical symbiotic structures (arbuscules and vesicles). Some studies as Vega et al. (2009) found high levels of AM colonization in Vaccinium plants cropped with the native populations of AM endophytes. In the present study the structures visualized in V. corymbosum roots resembled to dark septate endophytes (DSE), characterized by the presence of microsclerotial structures on the epidermal root cells according to described by Jumpponen (2001). In this case, the DSE colonization can reduce the AM colonization (Jumpponen and Trappe, 1998) or produce a negative effect on the extramatrical phase of the AM fungi (Scervino et al., 2009). Further studies using molecular tools are needed to corroborate the presence and extension of AM colonization in V. corymbosum roots, either in the acclimatization phase as well as in field conditions.
As Figure 2 shows, plants co-inoculated with any saprobe fungi did not showed an manifest affect on the AM colonization (25 and 46% for any interaction). In the Figure 2A and 2F the dual inoculation with C. rigida increases the colonization of Gigaspora rosea or G. intraradices (34 and 35% respectively). The same values was observed in the Figure 2A to the dual inoculation with P. chrysosporium and G. rosea (34%); however, the dual inoculation with P. chrysosporium and G. viscosum produced a significant increase in the percentage of root colonization (46%; Figure 2D). These results suggest that P. chrysosporium inoculation can produce a synergistic effect on the root colonization by G. viscosum, which could be used as a biotechnological tool for improving the adaptation of Vaccinium plants at acclimatization phase. Similar effect was observed with C. rigida in interaction with G rosea or G. intraradices. The specific mechanisms implicated in the beneficial effect of saprophytic fungi exudates on AM fungal colonization of roots are very complex and still poorly understood (Aranda et al., 2009).
3.2 Plant growth parameters
Only few inoculation treatments produced a significant increase in the shoot and root biomass production compared to the control treatment (Table 1 and 2). Among the treatments including AM fungal inoculation, such corresponding to G. claroideum and G. intraradices produced the higher shoot and root bio-mass. Therefore, although it was not possible to demonstrate clearly the AM colonization, these results suggest that these AM fungi had a beneficial influence on plantlet growth.
On the other hand, the inoculation with the saprobe fungus T. harzianum resulted in a significant increase of shoot and root biomass. The inoculation with saprobe fungi P. chrysogenum and T. versicolor produced the highest increase in shoot production, but this trend was not observed at root level. The improvement in the growth of plants inoculated with saprobe fungi has been reported for several plant species, and has been attributed to the production of plant growth promoting substances and antimicrobial compounds (Arriagada et al., 2009b; Arriagada et al., 2010). In addition to, several strains of the genera Penicillium fungi have been described as efficient P-dissolving fungi (Silva Filho et al., 2002).
The shoot and root biomass of plantlets co-inoculated with both mycorrhizal and saprobe fungi showed that certain particular combinations also can stimulate plant growth significantly (Table 1 and 2). This suggests a synergetic interaction between both types of fungi, resulting in a beneficial effect on plant growth (Arriagada et al., 2010). Thus inoculation with G. viscosum + P chrysosporium, and G. intraradices + C. rigida resulted in a significant increase in the shoot and root biomass. Furthermore, the interaction between G. viscosum + T. harzianum; G. rosea + C. rigida; G. rosea + P chrysosporium; G. intraradices + T. harzianum; G. constrictum + P chrysogenum and G. constrictum + C. rigida resulted in a significant increase only in the shoot.
The root dry weight of V. corymbosum was improved by G. intraradices inoculated alone or together with C. rigida, T. versicolor or P. chrysogenum (Table 2). In this sense, some studies have shown that the transfer of carbohydrate to the AM fungi and colonized roots produce a redistribution of photosynthates in comparison with non-mycorrhizal ones, being in this case a stronger carbon sink (Bago et al., 2000). Furthermore, the increase in root dry weight may be related with an increase in phytohormone production by the AM fungal colonization, which stimulates the development of the root system (Román García, 2003).
3.3 Soil enzyme activity
Certain soil enzyme activities were determined to check the effect of inoculation with AM and saprobe fungi on the biochemical and biological properties in the V. corymbosum rhizosphere. In general, dehydrogenase enzyme activity is considered as an index of the total microbial activity of the soil (García-Izquierdo et al., 2003; Arriagada et al., 2009a), then a greater activity would imply a higher biological oxidation of the organic matter and the concomitant higher solubilization and availability of mineral nutrients for the plants. De-hydrogenase activity in the rhizosphere of V. corymbo-sum was increased by the inoculation with all AM and saprobe fungi, but the interaction between both type of fungi not showed a remarkable difference (Table 3). In this case, among the many factors which may affect the biological activity of the soil (e.g. pH, availability of nutrients, soil humidity etc.), some studies showed that a soil pH < 5 has a negative effect on the dehydrogenase activity levels (Ramos and Zúñiga, 2008). In fact, the soil pH here was 5.84. This could explain the response of some combined treatments which presented a similar dehydrogenase activity to the uninoculated control.
The β-glucosidase activity in the rhizosphere of V. corymbosum was slightly increased by the inoculation with all AM fungi, but this effect was more evident in treatments with G. claroideum; G. viscosum and G. intraradices (Table 4). In addition to, the inoculation with any saprobe fungi resulted in a similar increase of this activity in the rhizosphere. Therefore, although there were not significant differences evidence an increase of β-glucosidase activity, it may be suggested that the AM and saprobe fungi generate a higher demand and competition for nutrients (Arriagada et al., 2009a; Richardson et al., 2009).
Directed inoculation with specific AM fungi and saprobe fungi strains to the rhizosphere of V. corymbosum cv. 'Elliot' favored significantly the growth and development of the plantlets, thus demonstrating the beneficial effect produced by the incorporation of these plant growth promoting microorganisms during an early growth stage in the greenhouse, and allowing to reduce the acclimatization time. In this order, further studies will be focused to understand the nature of the mechanisms involved in the interactions between AM fungi, saprobe fungi and Ericaceae plants, to design strategies to improve responses in the co-inoculation with both type of fungi.
Financial support for this study was provided by the Fondo Nacional de Desarrollo Científico y Tecnológico, FONDECYT, project NT100752 and Dirección de Investigación Universidad de La Frontera.
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