RESEARCH WORK

Use of EcoMic® and the bioactive product Pectimorf® in the establishment of two forage species

Gertrudis Pentón¹, Inés Reynaldo², G. J. Martín¹, R. Rivera² y Katerine Oropesa¹

¹Estación Experimental de Pastos y Forrajes «Indio Hatuey» Central España Republicana, CP 44280, Matanzas, Cuba
²Instituto Nacional de Ciencias Agrícolas, Mayabeque, Cuba
E-mail: gertrudis.penton@indio.atenas.inf.cu

ABSTRACT

In order to evaluate the effect of EcoMic®, Pectimorf® and/or their combination on the establishment of the forage species Morus alba L. (mulberry) and Sorghum bicolor L. (sorghum), two trials were conducted. The first one was done with M. alba, inoculated or not with AMF, and the plant yield and survival variables were evaluated after 90 and 240 days. The experiment with S. bicolor was conducted for 30 days after planting and seed germination and yield variables were evaluated after 7, 15 and 30 days. The treatments were: control, inoculation with AMF, imbibition in Pectimorf, and their combination. A randomized block design was used, with three repetitions. Response of mulberry to the inoculation with AMF was obtained after 90 days of establishment, in terms of green weight of the rootlets and roots, which tripled the results of the control. After 240 days the inoculated plants doubled the yield of the control and there were no significant differences in survival, increase of edible biomass per leaf area unit, or relationship of the assimilative system per edible biomass unit. In sorghum, the combined use of Pectimorf® and EcoMic® produced a significant increase, as compared to the control, in seed germination 30 days after planting (89 vs 58%), effective height (4,04 vs 3,55 cm), stem base diameter (0,18 vs 0,15 cm), leaf blade area, number of open leaves (4 vs 3) and leaf length. The products EcoMic® and Pectimorf®, as well as their combination, are concluded to constitute an effective alternative for the establishment of the studied forage species.

Key words: Growth, Morus alba L., Sorghum bicolor L., yield.

INTRODUCTION

The use of biofertilizers and bioactive products is a common practice in current agriculture. In the case of biofertilizers based on arbuscular mycorrhizal fungi (AMF), they are acknowledged for their capacity to increase agricultural production sustainably and in an ecologically acceptable way (between 15 and 50%). Their benefits are related to:

• nutrition, because they increase the absorption capacity of the root system; they are particularly successful in tropical soils with deficiency of assimilable phosphorus;

• relations to water (they allow absorbing water at lower potentials than root hairs);

• soil structure (they improve the physical properties, increase aggregate formation);

• heavy metals (they increase tolerance); and

• root pathogens (they increase tolerance).

With regards to the bioactive product Pectimorf®, its active principle is pectin which is obtained as a byproduct of the citrus fruit industry. It is a low-cost product, and the advantages of its use are related to:

• germination;

• plant vigor and survival during the early stages of plant development (it has an stimulating effect on root growth);

• pathogen organisms (it stimulates defense organisms); and

• drought and salinity (inductor of tolerance mechanisms).

In spite of all the above-explained facts, in the livestock production sector EcoMic® and Pectimorf® are insufficiently utilized. Some studies with the commercial product based on AMF report about its use in plantations of Leucaena leucocephala (Cuesta et al., 2006; Reyes, 2006) and Stylosanthes guianensis (González et al., 2004), and in pastures such as Cynodon nlemfuensis, Brachiaria decumbens and Panicum maximum = Megalothyrsus maximum (Vieito et al., 2006). There are no references to the bioactive product.

Hence, the objective of this work was to evaluate the effect of the product EcoMic® (based on AMF), the bioactive product Pectimorf® and/or their combination, on the establishment of the forage species Morus alba L. (mulberry) and Sorghum bicolor L. (sorghum).

MATERIALS AND METHODS

Location. The study was conducted at the Experimental Station «Indio Hatuey» (22º 48'7" latitude north and 79º 32' y 2'' longitude west) located at 19,9 masl.

Soil and climate characteristics. The soil of the site is Ferralitic Red (Hernández et al., 2003), with good surface and internal drainage. The chemical composition was characterized by contents of Na, K, Ca and Mg which varied between 0,04 and 0,08; 0,10 and 0,14; 10,8 and 16,0 and 3,2 3,7 cmol/kg, respectively.

P varied between 13,0 and 22,0 ppm and OM between 1,50 and 2,22%, while pH was 6,1-6,3.

Experimental procedure. Two independent trials (per species) were conducted. In the case of mulberry, the study, under field conditions, was developed at two moments of the crop establishment stage: 90 and 240 days. The planting, by cuttings, was done at the end of the rainy season, in a 1,0 x 0,5 planting frame, with east-west orientation. The plots were formed by 48 plants in an area of 24 m².

The experiment with sorghum was conducted during the first 30 days since seeding, in Styrofoam trays divided into cubbyholes with a capacity of 144 cm³. The substratum used consisted in a mixture of soil and organic matter from earthworm humus, in a 2:1 ratio.

The product EcoMic® is the commercial support of AMF. The fungal strain used was Glomus fasciculatum (Hoi like), obtained from certificated inoculi, with a content of 20-25 spores/g of inoculant. The inoculation was carried out by the method of immersing the seeds or the lower end of the cuttings in a fluid paste (0,8 kg/L of H₂O), recommended by Rivera et al. (2003).

The bioactive product Pectimorf®, based on pectin-type oligosaccharins, was obtained at the Laboratory of Plant Physiology and Biochemistry of the National Institute of Agricultural Sciences (INCA) (Mayabeque province, Cuba) and applied at a rate of 10 mg/L, by the method of seed immersion.

Treatments:

• Mulberry trial

- Control

- Inoculation with AMF

• Sorghum trial

- Control

- Inoculation with AMF

- Imbibition with Pectimorf®

- Inoculation with AMF plus imbibition with Pectimorf®

Evaluated indicators

In the mulberry trial the following indicators were evaluated:

- Branch number (u), green weight of the rootlets (g), root green weight (g) and dry matter yield of leaves (g/plant) after 90 days of establishment of the crop.

- Survival (%), effective height (cm), number of branches (u), leaf area ratio (LAR dm²/g), net assimilation rate in the edible biomass (NAT, g/dm²/day), leaf dry matter yield (LDMY, g/plant) and edible biomass dry matter yield (EBDMY, g/plant) after 240 days of establishment of the crop.

In the sorghum trial germination (%), effective height (cm), stem base diameter (cm), open leaf number and leaf length (cm) were evaluated.

Experimental design. In both trials a randomized block design was used, with three repetitions.

Mathematical analysis. The results were processed through a general lineal model ANOVA, and the means were compared through Duncan's (1955) multiple comparison test, for a significance level of 0,05. The statistical pack used was Infostat, free version. The variables branch number, green weight of the rootlets and roots were transformed according to Öx.

RESULTS AND DISCUSSION

The biofertilizer EcoMic® (based on AMF) as well as the bioactive product Pectimorf® are known to exert their influence on plant species, mainly through roots and rootlets. This assigns it especial importance for the survival and development of crops during the establishment stage, because of the fact that plants with more developed, finer, well distributed roots, with higher proportion of absorbing hairs, attract more nutritional elements. In turn, the absorption rate is higher with the increase of carbohydrates in the root and favors the accumulation of the respiratory substratum, with the subsequent increase of energy supply.

The mutualist relationship between plant and fungus is related to the carbohydrate supply the former offers to the latter. According to Yamashita (1985), sucrose and starch are the most metabolizable stored carbohydrates in the mulberry stems.

It is known that during the regrowth and rooting of mulberry cuttings, the accumulated quantity of carbohydrates in the ligneous stems decreases rapidly regarding the new developing organs. In this regard, Yamashita (1985) observed that the changes of their content in the phloem were very small during the first two days and in the 12 days afterwards there was a drastic reduction (35 to 10 mg of sucrose/g of dry weight and from 50 to 10 mg of starch/g of dry weight). From such moment the sucrose content increased to 20 mg/g of
dry weight, and after the 20^th day it remained stable, which justifies the intense root growth of the mulberry cuttings inoculated with AMF (table 1).

The branch number did not differ among treatments (table 1) and the values were two and three branches as average. Noda et al. (2004) stated that since 30 days after planting mulberry in the rainy season, a large number of regrowths and branches in the plants can be observed. In this work the limited branch emission coincided with the meteorological conditions that characterized the dry season the crop went through since its 60^th day of establishment. Nevertheless, the response to biofertilization in the rootlets and root green weight (indicators in which the inoculated plants tripled the results of the control) was remarkable, as well as in the dry matter yield in the leaves, which was significantly higher. Regarding this, it is stated that plant species with lower capacity to absorb the soil phosphorus and micronutrients are the most dependent on arbuscular mycorrhizal fungi. Their high aptitude for mycorrhization is also related to root morphology, to the absence of fine roots and well developed root hairs, and to a high growth potential (Riera, 2002). Padma et al. (2000), when referring to the mycorrhizal symbiotic fungi that colonize the bark of mulberry roots, emphasized their important role in the efficiency of phosphorus mobilization and the availability of a group of micronutrients, as well as in plant growth and development.

The response of roots and leaves to the inoculation with AMF coincided with the results obtained by Tikader and Roy (1999), who observed in 10 exotic mulberry accessions in Bengal that rooting had a positive correlation to stem length, leaf number and weight, stem weight, total biomass weight, root weight, length, volume and number.

The results showed the agronomic and ecological importance of biofertilization with AMF for the establishment of forage species in Cuba, because it increased plant tolerance to stress conditions (table 2). Pulido (2002) and Medina et al. (2006) stated that in many plant species, the fungus-host symbiosis favored water absorption as a result of the increase of water conductivity and the faster recovery of turgescence in situations of hydric stress or drought. Reddy et al. (1998) reported a significant response in terms of growth in mycorrhized mulberry plants cv. S13; such result was later validated under semiarid production conditions (Reddy et al., 2000).

It should be indicated that M. alba is a fast-establishment plant, which can last between 8 and 12 months under Cuban conditions (Domínguez et al., 2001), and according to FAO (1990) it should grow freely during 270 days before starting its intensive exploitation. Such species shows high susceptibility to the presence of weeds, mainly in the rainy season, for which it is usually planted in September and October for its establishment during the dry season. At the beginning of the new rainy season, depending on the plantation status, the establishment cutting is performed. It should be considered that the amount of phytoplasma in the plant organs is highly related to the growth period. The studies conducted by Dai-Qun et al. (1998) proved that in the mulberry stem such indicator showed a significant increase in the rainy months; however, during the dry winter it was almost undetectable. In the leaves this situation behaved similarly and the maximum values were observed in August. In the roots the phytoplasma quantity was lower and there were no large changes between seasons.

The production of edible dry matter per plant (table 2) allowed estimating between 1,0 and 2,5 t of edible DM/ha in the control and in the inoculation with AMF, respectively, which exceeded the values reported by Fernández et al. (2002) during the establishment of four mulberry varieties on a slightly acid grayish Brown soil, with a rainfall accumulate of 1 100 mm (12% of the rainfall in the dry season).

The effect of the inoculation with AMF on the indicators of mulberry yield after 240 days of establishment coincides with the report by Setua et al. (1999) and Setua et al. (1999a) about the direct influence of mulberry inoculation with Glomus mosseae on the growth in height, number of leaves per plant, leaf area and content of leaf moisture, with an increase of the leaf area dry weight of 27,4%. In this work the inoculated plants doubled the control yield without significant differences in the increase of edible biomass per leaf area unit (fig. 1), which explains the fact that there were no important variations in the balance between photosynthesis and respiration. There was no remarkable difference either in the relation of the assimilative system per edible biomass unit. This suggests that changes in the morphological characteristics occur in the root system, which determines positive effects in the entire plant organism.

With regards to S. bicolor, the use of mycorrhizae also constitutes a priority. Its establishment demands the strict control of weed competition, which slows down the crop. On the other hand, the lack of irrigation or opportune rains reduces the water potential gradient between soil and roots. Without this water transport which allows absorbing ions, the roots would have to be longer and more finely divided to obtain the nutrients. Hence increasing its competitive capacity, from a higher root development and apical growth, has an essential importance.

Sorghum is a highly demanding crop of specific agrometeorological conditions, to prevent water excess or deficiency during the plant growth and development. Correa (2001) stated that the planting stage is related to soil temperature; at a lower temperature, losses are increased. Few plants are as assertive as sorghum with regards to the need for vital space. The roots of this crop type release toxins to the soil which prevent the development of invading plants. These allelopathic properties of sorghum are stronger than in other plants (Pérez et al., 2010). Yet, as the seedlings are weak and grow very slowly after germination, the effect of competition with weeds during the first 3 or 4 weeks after emergence can be devastating. It is the period in which the crop must be kept clean.

AMF and active compounds based on oligosaccharins have shown significant potential in the increase of vigor and survival of herbaceous species during the early vegetative development stages. The research conducted in rice for its cultivation in semiarid zones proved that the pretreatment of seeds with Pectimorf® solution also stimulated the mechanisms of tolerance to drought and salinity (Vázquez, 2008).

In Cuba Pectimorf® has been validated in cucumber, potato, garlic, sugarcane, rice, tomato and citrus fruits, among other crops (Terrero, 2010). On the other hand, according to the studies conducted by Veeraswamy et al. (1992), the interaction of mycorrhizal fungi and brassinosteroids (the latter are bioactive products), has a significant effect on sorghum production.

Figure 2 shows the effect by the combined effect of Pectimorf® and EcoMic® on seed germination, with significant differences from the control, which after seven days had not exceeded 40% germination, and after 30 days only reached 58%. In figure 3 the contrasting images between the control and the treatment with Pectimorf® show its significant effect on the growth of the aerial and underground plant fractions.

The treatment with Pectimorf® allowed doubling the intensity of apical and root growth in the first 15 days (fig. 4). Although no significant differences were found, there was a trend of the stem base diameter to be higher in the treatment with Pectimorf®.

Table 3 shows that the application of EcoMic and/or Pectimorf® was manifested in a stimulation of growth, which was referred by Naranjo (2005) for the case of tobacco.

Different events allow explaining such results. The stimulation of plant growth in the presence of arbuscular mycorrhizal fungi does not have to be necessarily related to the solubilization of the little mobile elements in the soil or present at low concentrations. Such mechanisms as the production of phytohormones, vitamins or aminoacids can be linked to the effects of these microorganisms (Rivera et al., 2003; Siquiera et al., 2010). With regards to pectic polysaccharides, they are absolutely responsible for the intercellular cohesion in the middle lamella of the cell wall (Taiz, cited by Anon, 2010).

In the early 80's, a large number of studies aimed at a new approach about the function of the cell wall of plants and the polymers that constitute it, as it is a natural reservoir of a group of signaling molecules known as oligosaccharins. They are soluble oligosaccharides produced by the partial degradation of the polymers that constitute the cell wall, and are biologically active at very low concentrations, which characterizes this group of biomolecules as a new hormonal hierarchy in the context of communication between plants and the environment (Aldington, cited by Anon, 2010). Thus, the cell wall could act as some kind of pseudogland or deposit of precursors of a type of regulating molecules which, once released, are capable of controlling different functions related to the growth, organogenesis and defense against pests and diseases (Darvill and Albersheim, cited by Anon, 2010). Pectin constitutes 0,25 and 4% of the composition of the grape seed and pulp, respectively, and it is one of the four fibrous compounds recognized in such organs (hemicellulose, cellulose, lignin and pectin).

The optimum Pectimorf® concentrations in culture media to obtain a satisfactory biological response vary between 5 and 20 mg/L, similar range as the one used for traditional hormones. This biorregulator has as additional advantages, its solubility in an aqueous medium which is used to prepare the culture media and its stability to sterilize the media (http://www.inca.edu.cuproductospdfpectimorf.pdf).

In general, the significant growth of the sorghum treated with EcoMic®, Pectimorf® and their combination, responds to the fact that on the one hand Pectimorf® influences the activation of cell division and the elongation of cell walls (Naranjo, 2005), and on the other hand, mycorrhizal symbiosis enhances the efficiency of nutrient supply to plants.

The products EcoMic® and Pectimorf®, and their combination, are concluded to constitute an effective alternative for the establishment of the forage species M. alba L. and S. bicolor L.