On-line version ISSN 0718-3429
Idesia vol.30 no.2 Arica Aug. 2012
Volumen 30, Nº 2. Páginas 75-83 IDESIA (Chile)
Effect of symbiosis in the production of melon seedlings with arbuscular mycorrhizal fungi
Efecto de la simbiosis con hongos micorríticos arbusculares en la producción de plantines de melón
Christian Santander1, 2*, Jorge Olave1, 2
1 Centro de Investigación y Desarrollo en Recursos Hídricos.
2 Universidad Arturo Prat, Casilla 121, Iquique, Chile.
This research was performed in a semi-controlled greenhouse of the "Estación Experimental Canchones", in which the evolution of the effect of the mycorrhizal fungi over the growth parameters, differentiation, biomass, stress indicators and biochemical indicators for the production of the horticultural seedlings; and the percentage of mycorrhization obtained was evaluated. Inodorous type honeydew melon was used as the plant model. The mycorrhizal fungus Glomus intraradices Schenk and Smith, commercially known as MYCOSYM TRITON, was used for the inoculation at the moment of sowing with doses of 0; 20; 40 and 80 spores per plant. A completely randomized design was used; analysis used a factorial analysis variance (ANOVA) with the a posteriori LSD, using the statistical software INFOSTAT and an alpha value of 0.05. The results obtained in the destructive sample on the 50th day after sowing showed that the treatment with 40 spores per plant produced maximum root biomass, low etiolation index, a high value of the proportion of dry root weight to dry shoot weight. However, the activity of both endogenous and induced enzymes of nitrate reductase measured in the leaves was unaffected. We conclude that the symbiotic association between the roots of the melon plants and the mycorrhizal species Glomus intraradices produced a greater root biomass and very favorable conditions for transplanting.
Key words: Glomus intraradices, arbuscular mycorrhiza, Cucumis melo, seedling.
Esta investigación fue realizada en un invernadero semicontrolado de la Estación Experimental Canchones, donde se evaluó el efecto de los hongos micorríticos sobre los parámetros de crecimiento, diferenciación, biomasa, indicadores de estrés y bioquímicos para la producción de plántulas hortícolas, así como también el porcentaje de micorrización obtenido. El material vegetal utilizado fue melón cv. Honeydew tipo Inodorus. El hongo micorrítico utilizado fue Glomus intraradices Schenk y Smith, nombre comercial MYCOSYM TRI-TON, el cual fue inoculado al momento de la siembra con las siguientes dosis 0; 20; 40 y 80 esporas por planta respectivamente. Se utilizó un diseño completamente aleatorizado, realizándose un análisis de varianza multifactorial (ANOVA) y para la separación de medias se empleó el test LSD, mediante el software estadístico INFOSTAT a un α = 0,05. Los resultados obtenidos en el muestreo destructivo a los 50 días de siembra determinaron que el tratamiento inoculado con 40 esporas por planta presentó una mayor producción de biomasa radical, menor índice de ahilamiento y una mayor relación peso seco raíz-peso seco vástago; sin embargo, la actividad de la enzima nitrato reductasa endógena e inducida medida en las hojas no fue afectada. En esta investigación se concluye que la asociación simbiótica entre las raíces de las plántulas de melón y las micorrizas de la especie Glomus intraradices determinó una mayor producción de biomasa radicular y una condición más favorable para el trasplante.
Palabras claves: Glomus intraradices, micorrizas arbusculares, Cucumis melo, semilleros.
Microbiota plays an important role in agriculture since it contributes to soil fertility, improving soil structure and biodiversity and has a real effect on plant development (Avis et al., 2008). Among these microorganisms are the symbiotic mycorrhizal fungi, which allow the plants to explore a greater useful surface of the soil by the production of external mycelia connected to the root system, increasing the absorption of nutrients and water, while the fungus receives carbonated compounds from photosynthesis which are necessary to complete its life cycle (Azcón-Aguilar y Barea, 1980; Harley y Smith, 1983; Pereira et al., 1999).
One of the main effects of mycorrhizae is the improvement in the nutritional state of the plants, increasing the capture of phosphorus, calcium, copper, sulfur, zinc and iron; Smith et al. (2001) confirmed the absorption of two nitrogenous forms N-NO3 and N-NH4+ from the soil solution and their transfer to the associated plants. Based on this information, the objective of this study was to evaluate the effect of arbuscular mycorrhizal fungi on the architecture, biomass production, stress tolerance and synthesis of nitrate reductase in seedlings of the Inodorus type honeydew melon.
Materials and Methods
The research was performed in a semi-controlled greenhouse of the Canchones Experimental Station. The trial used cv. Orange Flesh of Inodorus type honeydew melon, called "melon tuna" in Chile, and included a period of 50 days after sowing. At planting, the mycorrhizal fungus Glomus intraradices was inoculated using the commercial MYCOSYM Tri-Ton MYCOSYM International AG (Switzerland), which contains latent spores at a concentration of 150 spores per gram. Seedlings were grown in thermoforming plastic trays with 72 cells with a capacity of 43 cc each, on a table 40 cm above the ground. The substrate used was a mixture of peat:Perlite 70:30; seeds were sown homogeneously at a depth of 1 cm. From the emergence of the cotyledons to the first leaf plants were irrigated with water. After the formation of the first leaf we applied a 1/3 concentration of fertirrigation, after the second leaf 2/3, and after the formation of the second leaf with the complete concentration (Table 1). The nutritive solution was adjusted to a pH of 6.0-6.5; electrical conductivity of 1.6 dS m1, equivalent to a osmotic potential (Ψs) of 0,067 Mpa. pH and E.C. were measured every three days to adjust the frequency and time of irrigation.
Table 1. Fertirrigation solution (meq L1) used in honeydew melon (Inodorus) seedlings.
1 Steiner modified (Casas, 2005); solution adjusted for concentrations of Cl y Na+ present in salty irrigation water.
Inoculation was performed together with sowing. We evaluated four doses (4 treatments); 0; 20; 40 and 80 spores plant1, using each tray as a treatment; each cells was a pseudo-replicate (72 cells). We took destructive samples of five plants of each treatment 50 days after sowing. We determined the percentage of mycorrhization of the roots using the method of Phillips and Hayman (1970) with some modifications, and calculated the degree of mycorrhization using the method of Trouvelot (Trouvelot et al., 1986). We evaluated the physical parameters tissue differentiation, production of dry biomass (leaf, stem and root), stem diameter, plant height, etiolation index and leaf surface, and the stress indicators specific leaf surface (SLF) and the ratio root dry weight to stem dry weight (RDW/SDW).
The biochemical parameters evaluated were the enzymatic activities of endogenous and induced nitrate reductase. For endogenous activity we used the method of Bar-Akiva et al. (1970), adapted by Valenzuela et al. (1987) and determined induced activity using the method of Bar-Akiva and Sternbaum in 1966, modified by Bar-Akiva et al., (1970) and adapted by Valenzuela et al. (1987). Color intensity was measured at 540 nm in a SPECTRONIC model GENESYS 2 spectrophotometer. A calibration curve was constructed in the range of 0 to 4 µM, using NaNO2 1 mM; the results were expressed in µM NO2 * g1pf h1.
The experimental design was completely randomized. Analysis employed factorial ANOVA with LSD a posteriori tests, using the software INFOSTAT and α = 0.05. The number of leaves was log transformed using log10(X+1); percentage values were transformed with the Arcsine Transformation.
Results and Discussion
Percentage of mycorrhization
Inoculation with different doses of G. intraradices spores in melon seedlings produced significant differences in the percentage of mycorrhization (Figure 1). The greatest percentage was observed with 40 spores plant1 (T2, 49.8%), followed by T3 (80 spores plant1) with 36.3% and T1 (20 spores plant1) with 16.9%. The mycorrhization response with different doses of G. intraradices reached a maximum and then decreased (Figure 2), indicating that there is an optimum concentration of G. intraradices near 40 spores plant1, and that greater concentrations do not increase the percentage of mycorrhization. Similar results were obtained in tomato cv. Mara by Mujica and Medina (2008), in which there was no further increase after doubling the dose of Glomus mosseae and Glomus Hoi-like. Anaya et al. (2009) also obtained the best response in tomato seedlings inoculated with EcoMic (Glomus fasciculatum) at a medium dose.
Figure 1. Percent of mycorrhization in roots of Inodorus var. Honeydew melon 50 days after inoculation with G. intraradices. Different letters indicate significant differences (LSD, p < 0.05).
Figure 2. Effect of the dose of spores of G. intraradices on the percentage of mycorrhizal colonization in roots of melon Inodorus var. Honeydew 50 days after sowing.
Wang et al. (2010), obtained results similar to those reported here with treatment T2, they inoculated seedlings of melon cv. 901 and 908 with 10 g of substrate containing spores of different mycorrhizae, obtaining after 45 days 39.89% for Glomus versi forme and 51.75% for Glomus mosseae (cv. 901) and 46.38% for G. versiforme and 57.08% for G. mosseae (cv. 908); there were different percentages of mycorrhization between both fungal species and melon cultivars. Contrasting results were reported by Huang et al. (2011), who found 70% mycorrhization in seedlings of cv. Zhongmi 3 melon inoculated with G. intraradices, and Martínez-Medina et al. (2011), who reported only 8% mycorrhization of seedlings of cv. Giotto inoculated with Glomus intraradices.
The highest percentage obtained in this study inoculating cv. Honeydew melon with Glomus intraradices was 49% (T2), which shows that even different cultivars of a species have different degrees of mycorrhization when inoculated with the same fungal species. This agrees with the results of Wang et al. (2010), indicating that the success of plant symbiosis is based on the adequate establishment, development and extension of symbiotic mycorrhizal; this depends on the fungal species and its genetic characteristics, the environment in which it was isolated and its affinity with the plant species and/or cultivar. Other factors which influence symbiosis include the physiology of the plants and the properties of their roots (Brundrett, 2002, Drew et al. 2006); these authors reported that the mycorrhizal colonization process is determined by the capacity of soil exploration, which is greater in Glomus mosseae than in G. intraradices.
Growth, differentiation and biochemical parameters
Inoculation with G. intraradices did not affect the production of leaf biomass, stem or total biomass (Table 2). The main effect was produced in the production of dry root biomass; the differences among treatments were highly significant (p = 0.0001); treatment T2 had the greatest dry weight with 0.21 g, 31.25% and 75% greater than T3 and T1, respectively, and 133% greater than the control. The differences in the production of total dry mass were not significant.
Table 2. Production of dry biomass in Inodorus var. Honeydew melon seedlings 50 days after sowing, inoculated with G. intraradices.
LDW: leaf dry weight; SDW: stem dry weight; RDW: Roots dry weight; TDW: total dry weight. ns: not significant. Means in the same column with different letters indicate significant differences (LSD, p < 0 .05).
Mycorrhization of melon seedlings with Glomus intraradices produced a descending movement of photosynthates, coinciding with Wu et al. (2006) who reported that the mycorrhizal fungus generated a basipetal movement of photosynthates towards the roots. This movement is generated principally by the interchange of nutrients between the fungus and the plant; the hyphae of the fungus increase the effective root system and provide the plant with nutrients outside the depletion zone, while the plant transports carbohydrates towards the root which provide a substrate for the growth and development of the fungus. It has been observed that the greater the level of mycorrhization, the greater the fungal demands for substrate, producing greater movement of photosynthates.
The mycorrhization of the seedlings stimulated root growth; there was 4% more accumulation of dry material compared to the control, coinciding with the results of González-Monterrubio et al. (2005), who reported a significant increase of 59.2% in dry root biomass of Opuntia streptacantha seedlings inoculated with mycorrhizal fungi, and with those Huang et al. (2011), who obtained a significant increase by inoculating melon seedlings.
Mycorrhization produced more vigorous plants after transplanting, confirming the proposal for Tessi (1991) and Hoyos (1996), who proposed that greater accumulation of dry material in seedlings makes them more resistant to transplant. Tessi (1991) suggested that a 1% increase in seedling dry material increases the percentage of rooting by 30%, while Oseni et al. (2010), proposed that an increase in root weight in plants with mycorrhizae produces a positive correlation with nutrient absorption and a better post-transplant response, expressed as greater plant growth.
Parameters of growth and differentiation
The only significant difference in leaf differentiation and growth parameters was in the etiolation index (p = 0.0340). The melon seedlings inoculated with 40 spores plant1 had a more equilibrated distribution between height and diameter (IA: 25.89) compared to seedlings inoculated with 20 and 80 spores plant1 and the control, which had 6%, 26% and 52% more disequilibrium, respectively. Although there were no significant differences in the majority
of the parameters measured, the plants inoculated with 40 spores*plant1 had the best response in the parameters of growth and differentiation, except for plant height (Table 3).
Table 3. Growth parameters in seedlings of melon Inodorus var. Honeydew at 50 days of planting, inoculated with G. intraradices.
SD: Stem diameter; PH: Plant height; EI: Etiolation index (PH/SD); LS: Leaf surface; ns: not significant. Means in the same column with different letters indicate significant differences (LSD, p < 0 .05).
Although the differences in number of leaves, leaf surface and stem diameter were not significant, mycorrhization appeared to stimulate the growth of these parameters. An increase in stem diameter was observed in plants of Poncirus trifoliata (Wu et al., 2010), Cucumis melo (Huang et al., 2011), Leucaena leucocephala (Flores-Bello et al., 2008), Lycopersicum esculentum (He et al., 2010) and Calocedrus decurrens (Amaranthus and Steinfeld, 2005) when inoculated with mycorrhizae. Preciado et al. (2002) suggested that stem diameter is a good indicator of plant vigor, since it reflects directly the accumulation of photosynthates which after transplantation can be translocated to the demand sites. This was confirmed by Ortiz et al. (2009), who indicated that a thicker stem implies greater phloem area and thus more efficient transport and a greater reserve capacity of photosynthates. Mycorrhization generated a lower etiolation index, favoring better plant architecture; these characteristics are desirable to obtain seedlings with a better capacity to support transplant (Preciado et al. 2002).
Indicators of stress
The stress indicator most sensitive to mycorrhizal activity was the relation RDW/SDW and not SLF, since the former considers the behavior of the root where the symbiotic action occurs. Plants inoculated with Glomus intraradices had less stress than those not inoculated, since they had a greater RDW/SDW. The plants inoculated with 40 spores plant1 showed less stress (0.33) than those inoculated with 20 or 80 spores*plant1, due to greater root weight (Table 4).
Table 4. Indicators of stress in melon seedlings Inodorus var. Honeydew inoculated with G. intraradices 50 days after sowing.
SLS: Specific leaf surface (LS/LDW); R (LDW/SDW): (Root dry weight/Stem dry weight); ns: not significant. Means in the same column with different letters indicate significant differences (LSD, p < 0.05).
Our results are coincident with those obtained by Tobar et al. (1994) in lettuce, Meddad-Hamza et al. (2010) in micropropagated olives, Lee and Kim (2004) in cucumber and Oseni et al (2010) in tomato; these authors all reported an increase in the ratio RDW/SDW in plants with mycorrhizae, showing a high degree of efficiency of these fungi. This indicates that seedlings with mycorrhizae had a better value for this stress indicator and a better condition to support transplant, since they produced plants with better equilibrium between plant height and stem diameter.
Enzymatic activity of nitrate reductase
The enzymatic activity of nitrate reductase in leaves was not affected by the activity of Glomus intraradices in the inoculated plants (Table 5). All treatments received 7.5 meq L1 NO31 in the fertirrigation solution; however, there was a better NRI/ NRE ratio (Induced Nitrate Reductasa/Endogenous Nitrate Reductase) (T1:18; T2:24 y T3:39%), which indicates a greater availability of NO31 in the leaf tissue which may be reduced by the enzyme nitrogen reductase compared to the control.
Table 5. Nitrate reductase activity in seedlings of melon Inodorus var. Honeydew inoculated with G. intraradices at 50 days of sowing.
NRE: Endogenous Nitrate Reductase; NRI: Induced Nitrate Reductasa. ns: not significant. Means in the same column with different letters indicate significant differences (LSD, p < 0 .05).
Figure 3. Comparison between the plants without inoculation (left) and with treatment T2 (right).
This indicates a certain level of abiotic stress present in seedlings with mycorrhizae, and concurs with the proposal of Tobar et al. (1994). The greater availability of NO31 in plants with mycorrhizae is explained because part of the nitrate was absorbed in the soil by the external mycelia of the fungi and reduced (Bago et al. 1996 y Johansen et al. 1996), allowing the formation of intermediate molecules of the nitrogen cycle which are transferred to the plant for protein synthesis (Tilak and Dwivedi 1990, Subramanian and Charest 1998).
Figure 4. Fungal structures of Glomus intraradices colonizing the roots, staining with Tripan blue. A: Non-mycorrhized roots T0 (40X); B: Mycorrhized roots T2 (40X); C: Spores (100X); D: Vesicles (100X); E: Arubuscles (100X); F: Intraradical mycelium (100X).
The inoculation at sowing with Glomus intraradices produced more equilibrated seedlings of honeydew melon, with a greater root development than non-inoculated plants, a lower etiolation index and greater availability of nitrates in the leaves for reduction by nitrate reductase. The dose of 40 spores*plant1 produced the greatest percentage of mycorrhization (49%), a lower level of stress due to greater production of root biomass and 24% more nitrogen available for reduction in the leaves.
Centro de Investigación Avanzada en Recursos Hídricos y Sistemas Acuosos (CIDERH) CONICYTREGIONAL R09I1001. Departamento de Agricultura del Desierto y Biotecnología of the Universidad Arturo Prat State of Chile. Bio Triton Chile, for their support of this research by supplying the product MYCOSYM Tri-Ton.
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Fecha de Recepción: 26 Mayo, 2012. Fecha de Aceptación: 10 Junio, 2012.
* Correspondencia a: Christian.Santander@ciderh.cl