RESEARCH WORK

Effect of the mycorrhizal symbiosis, chemical fertilization and their combination, on the soil-plant relation of mulberry

Gertrudis Pentón¹, R. Rivera², G. J. Martín¹, Aracelis Mena², F. Alonso¹and Aida Medina ²

¹ Estación Experimental de Pastos y Forrajes Indio Hatuey, Universidad de Matanzas Camilo Cienfuegos, Ministerio de Educación Superior. Central España Republicana, CP 44280, Matanzas, Cuba.
²Instituto Nacional de Ciencias Agrícolas, MES, Mayabeque, Cuba
E-mail: gertrudis@ihatuey.cu

ABSTRACT

The effect of the inoculation with arbuscular mycorhizal fungi (AMF) strain Glomus cubense through Canavalia ensiformis, chemical fertilization and their combination, on the root functioning, mycorrhizal symbiosis and the nitrogen extraction capacity in mulberry (Morus alba L.), was evaluated. The fertilizer doses were: 1) control without fertilization (F0); 2) 100-50-50 kg of N, P and K ha^-1 per season (F1); and 3) 200-100-100 kg of N, P and K ha^-1 per season (F2). The alternatives to inoculate AMF consisted in: control without AMF, direct application to the soil, and inoculation through intercropped C. ensiformis. The root biomass of mulberry when inoculating AMF through C. ensiformis was higher (490,28 mg 50 g^-1 of soil). The mycorhizal symbiosis 150 days after the treatments were applied was higher with AMF inoculated through intercropped C. ensiformis and complemented with F1 (375,33 spores in 50 g of soil and 1,46 % of visual density). The nitrogen extraction through the edible biomass, in the treatment of AMF inoculated via C. ensiformis with F1, varied between 245,68 and 336,75 kg ha^-1 in the rainy season and between 74,59 and 97,81 kg ha^-1 in the dry season. It is concluded that the highest distribution of roots in the soil, the best indicators of mycorhizal symbiosis and the highest nitrogen extractions of mulberry were positively associated to the inoculation with AMF through C. ensiformis, complemented with chemical fertilization.

Key words: Biomass, Canavalia ensiformis, fungi, inoculation.

In current agriculture it is necessary to adopt strategies to stimulate and optimize soil biological processes, which favor nutrient recycling and give priority to the adoption of multifunctional techniques that maintain or improve fertility, counteract the erosion processes and promote the presence of populations of beneficial organisms.

In such context, arbuscular mycorhizal fungi (AMF) stand out within the wide range of soil microorganisms that are used in the production of biofertilizers (Calderón and González, 2007), whose action in the soil-root interface allows to reduce the applications of mineral fertilizers. For such reason, their use in intensive exploitation systems of mulberry (Morus alba) for forage production (Martín et al., 2002; Villalba, 2009; Oropesa et al., 2011; Noda and Castañeda, 2012) could be an important alternative to reduce the high quantities of these inputs under the current conditions.

Plant inoculation with mycorhizal fungi induces the increase of the absorption and relocation processes of nutrients, such as: P, N, K, Ca, Mg, Zn, Cu, Mo and B (Siqueira et al., 2010). Its effectiveness is guaranteed with management practices that stimulate the multiplication of infective propagules (González et al., 2008).

The use of Canavalia ensiformis as inoculation means of AMF constitutes a way for the biofertilization of established mulberry plantations, which in turn can be complemented with different fertilization sources and forms (Martín, 2009).

Due to all the above-explained facts, the objective of this research was to evaluate the effect of AMF inoculation through C. ensiformis, mineral fertilization and their combination, on some indicators of the soil-plant relation in an established mulberry plantation.

MATERIALS AND METHODS

Geographical location. The study was conducted in areas of the Pasture and Forage Research Station Indio Hatuey, Perico municipality Matanzas province, Cuba , located between 22º 48' 7" North latitude and 81º 2' West longitude, at 19,01 m.a.s.l. (Academia de Ciencias de Cuba, 1989).

Edaphoclimatic characteristics. During the experimental period (November, 2008-November, 2010) the rainfall showed variability, with annual accumulated values between 1 089 and 1 860 mm (table 1). The dry season had 18,77 % of the rainfall. The average temperature values oscillated between 21,07 ºC in the dry season and 26,04 ºC in the rainy season.

The soil has plain topography and is classified as hydrated Ferralitic Red (Hernández et al., 2003), with good surface and internal drainage. The chemical composition is shown in table 2.

Origin and characteristics of the genetic resources used. The plant species used in the study were: M. alba L. var. tigreada, as main crop, and C. ensiformis as a multiplication means of AMF. The commercial product used to inoculate the AMF was EcoMic®, strain Glomus cubense (Fernández et al., 2001).

Procedure. The mulberry was four year old at the beginning of the experiment. The sowing frame was 26 666 plants ha^-1, with a double-row spatial arrangement (1,0 x 0,50 x 0,50 m). The plot size was 24 m² and the rows were East-West oriented. The criterion of not applying irrigation was assumed, according to the real situation of most commercial farming exploitations. During the first 15 days of May and November, according to the established treatment, chemical fertilization was manually applied, locally per plant. Afterwards, inoculation with AMF was performed directly to the soil or through C. ensiformis seeds, planted between the mulberry rows, at a distance between plants of 40 cm and 50 cm away from the mulberry rows.

Ninety days after the beginning of the dry season and 60 days after the start of the rainy season, the C. ensiformis plants were cut and they were mulched on the mulberry row.

Chemical fertilization. Three doses of chemical fertilizer (F) were used: 1) control without fertilization (F0); 2) 100-50-50 kg of N, P and K ha^-1 per season (F1); and 3) 200-100-100 kg of N, P and K ha^-1 per season (F2). The fertilizer sources were: urea, simple superphosphate and potassium chloride.

In addition, the soil profile was analyzed in the variable root density. The evaluated depths were: 0-20, 20-40, 40-60 and 60-80 cm.

Experimental design. The design consisted in four randomized blocks, with 3 x 2 factorial arrangement.

Treatments established in the bifactorial analysis

F0: without AMF or fertilization

CeAMFF0: AMF with intercropped C. ensiformis and without fertilization

F1: 100-50-50 kg of N, P and K ha^-1 per season

CeAMFF1: AMF via C. ensiformis, complemented with 100-50-50 kg of N, P y K ha^-1 per season

F2: 200-100-100 kg of N, P and K ha^-1per season

CeAMFF2: AMF via C. ensiformis, complemented with 200-100-100 kg of N, P and K ha^-1 per season

Evaluated indicators

Root density (expressed in milligrams per every 50 g of soil) of mulberry in the soil profile, every 20 cm and up to 80 cm of depth, at the end of the experimental period. It was determined from the extraction of 25 x 25 x 20 cm soil blocks, at both sides of the selected plants, which formed 1/3 of the soil volume corresponding to the vital surface of the crop up to 80 cm of depth. The root system formed by roots and rootlets was wet sieved, and it was weighed on dry basis. The sample size was two plants per plot.

Spore count at 20 cm of depth (number of spores per 50 g of soil). During two years, in treatments CeAMFF0 and F0, the multiplication dynamics of infective structures (spores) at 15 days, after the first and second cutting of the dry season and the first cutting of the rainy season was studied. Besides, it was evaluated at the end of the experimental period, in the rainy season, 150 days after the treatments were applied. The procedure for the evaluation consisted in taking soil samples of 50 g, which were wet sieved and decanted from the fungus propagules, according to the extraction method described by Gerdeman and Nicholson (1963). The spores were collected on a mesh of 40 µm-holes. Afterwards, they were separated by centrifugation with sucrose and Tween 80, and they were observed in an optical stereomicroscope (20-40 x). The sample was two plants per plot.

Fungal colonization (%) and visual density of the colonization (%) at 20 cm of depth were determined at the end of the experimental period, in the rainy season, 150 days after the treatments were applied. For such purpose, the plant roots collected up to 20 cm of depth were screened. Then, they were washed with tap water to eliminate all the soil and were air-dried. Approximately 200 mg of rootlets were weighed, which were dried at 70 ºC and were tinted according to the method described by Phillips and Hayman (1970). The evaluation was made following the method of intercepts, developed by Giovanetti and Mosse (1980), to determine the percentage of mycorhizal colonization or the colonization frequency, from the formula:

The visual density percentage (% VD) was determined through the method proposed by Trouvelot et al. (1986), through which the fungal occupation of each intercept was evaluated and a level was assigned to it, being described below:

% of visual density = A / Z

A =Σ No. of intercepts in each level due to its points, and Z = Σ (0-5).

Where:

Z: number of intercepts counted in each level.

A: result of the multiplication of Z by the observed occupation percentage.

The evaluation of the indicators of mycorhizal symbiosis was made at the end of the rainy season, 150 days after the inoculation with AMF, because in this season the mycotrophic performance of mulberry is better expressed. In addition, 150 days were considered as a period that allows to evaluate the permanence of infective propagules in the soil and the efficiency of colonization in a date previous to the re-inoculation

Nitrogen extraction through the edible biomass of mulberry. Nitrogen (N) extraction was calculated during two years (kg ha^-1 per season), from the data of the dry mass and its corresponding concentration, by means of the formula:

Extraction of N = [Dry mass x % of N in leaves and fresh stems] / 100

Statistical processing. The analysis of the results was made per season, taking into consideration the results obtained by Martín (2004), who proved the seasonality of mulberry. The suppositions of normality of the errors, as well as the variance homogeneity, were tested. Likewise, the variables were transformed into √x for the percentage data (fungal colonization and visual density) or log x for the number of AMF spores. A linear general model was used which included the studied effects and their interactions

( Y_ijk= μ + F_i+ inoculation of AMF_j+ block_k+ e_ijkl.).

For mean comparison Duncan's (1995) test was used, for pd» 0,05. The statistical pack used was InfoStat free version (InfoStat, 2004).

RESULTS AND DISCUSSION

Figures 1 and 2 show the significant effect (p ≤ 0,05) of the inoculation alternatives and the soil profile, as independent factors on root density. The alternatives with AMF inoculated through intercropped C. ensiformis were remarkably higher than F0 and F1. There was a trend to increase in the volume of roots as the inoculation of AMF was complemented with fertilizer, although without significant differences. It is based on the fact that the fungus hyphae capture the simple nitrogen from the decomposing organic matter, of the soil, and facilitate the access to the available macro-and micronutrients, stimulating plant growth and, in the presence of fertilizer, propitiating an intense root growth (Hodge, 2002).

In treatment CeAMFF0, the absence of chemical fertilization should have been compensated by the absorption of mineral elements through the AMF structures, which demanded higher root growth. This was also stimulated by the presence of C. ensiformis (sown at low density). Entry et al. (2002) stated that roots inoculated with mycorhizae are capable of obtaining more nutrients than the ones that are not, because hyphae explore a higher soil volume. This is very important under the conditions of this study, as the content of organic matter in the soil was moderate, and the exchangeable potassium, low. Paneque and Calaña (2001) considered that the values lower than 0,15 or between 0,15 and 0,30 cmol kg^-1 of potassium indicate low presence of this cation in the soil. Regarding the organic matter content, the authors qualified 3 % of OM as moderate.

Regarding the incidence of the soil profile on root density (fig. 2), the trend was to decrease in the deepest layers. The average root volume was significantly higher in 0-20 cm, and there were no remarkable differences in 20-40 and 40-60 cm, or in 40-60 and 60-80 cm.

It is known that root density appears in typical gradients between the surface soil layer and the subsoil. The high root density in the surface layers is mainly caused by more favorable physical, chemical and biological conditions, and the logarithm of root density linearly decreases with the increase of depth (Rivera, 1987).

It should be acknowledged that, in spite of the lower root density in the subsoil, the absorption of nutrients from this zone could be considerable. The absorption from the subsoil is also significant for mineral nutrients, such as: ammonium nitrate, magnesium, potassium and phosphorus. The relative importance of the supply from the subsoil depends, among other aspects, on the root density in this zone (Hernández, 2001).

Figure 3 describes the multiplication dynamics of AMF structures between 0 and 20 cm. The content of native propagules in the rhizosphere varied between 75 and 434 spores in 50 g of soil, in treatment F0, which is considered from moderate to low according to the criteria expressed by Rivera and Fernández (2003). These authors classify the density as low when it is below 200 spores in 50 g of soil; moderate, from 300 to 500; and high, when it exceeds 600 spores in 50 g of soil. Such criteria allow to consider treatment CeAMFF0 as effective, because in almost all the cuttings the number of infective structures was higher than 400 spores in 50 g of soil, with relevance in the first cutting of the rainy season of the first year, which reached values of 917. In this sense, the difference with regards to F0 was remarkable and it also constitutes an indicator that mulberry is a mycotrophic species.

The decrease of the population of spores in February of the first year was remarkable, in F0 as well as in CeAMFF0, which showed the limitations in the multiplication capacity of infective structures during the driest months.

In this regard, it is known that AMF are obligatory symbionts and that their distribution in cultivated soils is strongly influenced by the vegetation, climate variables and soil characteristics. The population of AMF spores is significantly raised with the increase of the number of plant species present in an agricultural system (Chen et al., 2004), in contrast with monocrop mulberry plantations. Peña et al. (2006), when analyzing the appearance frequency of native AMF spores per type of cover, found that the agroforestry systems, followed by the natural forest, showed the highest averages of root colonization (over 30 %). On the contrary, the covers of land plots, as well as the monocrop and the grazing areas showed the lowest values of root colonization. In other studies, these authors proved that the arbuscular mycorhizal symbiosis was more effective in ecosystems with highly heterogeneous covers; hence the importance of promoting biodiverse agricultural production systems (Peña et al., 2007), which minimize the fragility of monocrop plantations, as in the case of mulberry.

Table 3 shows the results of the multiplication of infective propagules at the moment of inoculation and 150 days after the treatments were applied. The content of native propagules in the rhizosphere (F0) was low.

There was a significant effect of all the treatments on spore multiplication 150 days after being applied, in the rainy season. The visual density of the mulberry roots was remarkably higher in the treatment CeAMFF1.

The highest visual density and the colonization in F1, compared with the maximum dose (F2), confirm the report by Rivera and Fernández (2003) about the negative effect of the high doses of chemical fertilizer on the mycorrhizal symbiosis. Likewise, the decrease of its effectiveness is a consequence of the malfunctioning of the structures or of the inhibition of symbiosis.

The high phosphorus supply can cause an unbalance among this element, nitrogen and potassium, which stimulates root growth but inhibits fungal functioning. On the contrary, if the phosphorus availability is low or null, the symbiosis does not work correctly, and the effectiveness of inoculation is low (Mohammad et al., 2004). It is estimated that the association between the host and AMF consumes between 5 and 10 % of the products of photosynthesis. This cost is compensated if the plant is under suboptimal conditions of nutrient supply (Siqueira et al., 2010).

The colonization values below 25 %, 150 days after the beginning of the rainy season, indicated the need to artificially introduce AMF, through inoculation at the start and at the end; and thus increase the effects of symbiosis, in the rainy as well as in the dry season.

In this sense, in several works the influence of soil type and seasonality on mycorrhizal functioning has been proven. In soils with good drainage, the highest colonization percentages were found in the rainy season (Apple et al., 2005; De Oliveira and de Oliveira, 2005; Becerra et al., 2007); while as the soils were less fertile a higher quantity of fungal structures became necessary, to guarantee the adequate functioning of symbiosis (Azcón et al., 2003).

Regarding the nitrogen extractions through the edible biomass of mulberry (table 4), the highest ones were observed to coincide with the treatments of higher nutrient contribution. The highest values oscillated between 245,68 and 336,75 kg ha^-1 of N in the rainy season and between 80,63 and 108,08 kg ha^-1 of N in the dry season. The treatments CeAMFF1, F2 and CeAMFF2 did not differ among themselves in the first year of evaluation and were significantly higher than the others. In the second year the highest extractions were obtained in CeAMFF1.

The results proved the convenience of the inoculation of AMF through intercropped C. ensiformis, complemented with fertilizer doses below the optimum level, since the extracted nitrogen volume under this condition did not differ from the maximum dose. In addition, fertilization was more efficient.

In general, the high extractive capacity of mulberry became evident in all the treatments, which is in correspondence with international literature. In this sense, some data compiled by Ito and Takagishi (1990) proved that in mulberry plantations yielding 24,8 t ha^-1 of leaf biomass per year, the total extracted nitrogen from the soil was 242 kg ha^-1 per year; from them, 206 kg ha^-1 per year were exported from the plantation through the leaves.

CONCLUSIONS

The use of C. ensiformis as multiplication means of infective AMF propagules, in mulberry plantations, is feasible.

The highest distribution of M. alba L. roots in the soil was positively associated to the inoculation of AMF through C. ensiformis, complemented with chemical fertilization.

Mulberry has mycotrophic character, and the mycorrhizal symbiosis is effective after the treatments have been applied in the rainy season.

It is necessary to fertilize with NPK, at a rate of 100-50-50 kg ha^-1 per season, at the beginning and end of the rainy season.

Under the above-explained condition a close soil-plant relation was guaranteed through the root growth, which favored mycorrhizal symbiosis as indicator of the mycotrophic functioning status of the crop and soil, and the high nitrogen extractions as the crop response to the productive capacity of the soil.

Received: June 10, 2013
Accepted: June 20, 2014