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Using Trichoderma viride to optimize earthworm composting process, improve earthworm composting quality and prolong storage period

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https://www.eduzhai.net/ International Journal of Agriculture and Forestry 2014, 4(5): 343-350 DOI: 10.5923/j.ijaf.20140405.01 Using Trichoderma viride for Optimization of Vermicomposting Processes to Improve the Quality of Vermicompost and Prolong the Storage Period Natalia N. Tereshchenko*, Alla B. Bubina, Tatiana V. Yunusova Siberian Institute of Agriculture and Peat, Siberian Branch, Russian Academy of Agricultural Sciences, Tomsk, 634050 Russia Abstract The performed study demonstrates that a preliminary fermentation of organic substrate with Trichoderma is promising, since this allows the vermicultivation time to be reduced and the yield and quality of vermicompost to be elevated. Vermicompost obtained after pre-fermentation of organic substrate with Trichoderma has the maximal indicators of growth-stimulating activity and the highest level of fungistatic properties in relation to root rot pathogens. This variant of vermicompost maintains the highest level of biological activity even after 8 months of storage. During the storage period growth-stimulating activity of vermicompost changes cyclically. The level of growth promoting activity of vermicompost is greatly influenced not only by period of storage, but also by the stage of organic matter transformation in vermicompost at the end of the process of vermicultivation. Excessive prolongation of vermicultivation causes a decrease in its biological activity. Keywords Vermicompost, Vermicultivation, Trichoderma viride, Introduction, Growth-stimulating activity, Fungistatic activity, Storage period 1. Introduction The efficiency of introduction of the active fungal strains belonging to the genus Trichoderma into the plant rhizosphere, as well as preplanting inoculation, has been demonstrated in numerous studies [1–3]. Despite the fact that preparations involving Trichoderma as the major component is a real alternative to synthetic agrichemicals as antagonists of plant soil diseases and growth stimulators, their commercial application presents certain difficulties. Application of such preparations does not always give stable results, especially under field conditions [4]. Ecological studies have shown that fungal strains belonging to the genus Trichoderma can not always be successfully introduced into the soil microbial community due to their low competitiveness and high demand for available sources of carbon. For this reason, their influence as biological control agents is limited to a few weeks after introduction [5]. Besides some technological problems caused by short shelf life of the liquid form of the preparations based on Trichoderma frequently take place. Whereas the use of the solid preparation form often causes the problem of autoinhibition of conidia germination. For this reason, there is a need for a careful calculation of the optimal dose for each * Corresponding author: ternat@mail.ru (Natalia N. Tereshchenko) Published online at https://www.eduzhai.net Copyright © 2014 Scientific & Academic Publishing. All Rights Reserved particular strain [6]. Therefore, development of multifunctional biological preparations stable in their efficiency, including the preparations involving Trichoderma, is among the most important tasks in the system of developing organic crop farming. In order to maintain a stable population of Trichoderma in the soil during the growing season, some authors propose to perform the application of Trichoderma simultaneously with the introduction of various organic substrates: wheat bran, peat moss, pine bark, compost from cow manure and even waste substrates after oyster culturing [7, 8]. In our opinion, a joint application of Trichoderma and vermicompost is promising technological method to raise Trichoderma survival in the soil and fungal colonization of the plant rhizosphere. On the other hand, since the quality of vermicompost depends on the content and activity of the agronomically valuable microflora, vermicompost enrichment for the active microbial strains responsible for fungistatic, growth-stimulating, and other beneficial properties can have a positive effect on the efficiency of its use. It is also known that many species of the genus Trichoderma are able to accelerate the composting rate of organic substrates due to their ability to transform the lignin–cellulose complex with their cellulolytic enzymes, which, in turn, enriches the final product for nutrients [9]. In accordance with the above arguments the goal of this work was to improve the technology for producing vermicompost by preliminary fermentation of organic 344 Natalia N. Tereshchenko et al.: Using Trichoderma viride for Optimization of Vermicomposting Processes to Improve the Quality of Vermicompost and Prolong the Storage Period substrate using the fungus Trichoderma viride with the aim to reduce the time required for vermicultivation and to elevate the growth-stimulating and fungistatic activity of vermicompost. 2. Experimental Objects of study were the earthworm Eisenia andrei and a pure culture of the saprophytic fungus Trichoderma viride, isolated by the authors from pine wood. The growth-stimulating activity of vermicompost was determined by biological tests and vegetation experiments with the wheat cultivar Tulunskaya 12. The earthworms were cultivated in a peat–manure mixture (PMM) composed of fen peat from the Kandinskoe deposit and cattle litter manure at a ratio of 1: 4 (wt/wt) with 40 mature earthworm individuals (30 g) added per 1 kg substrate. The moisture content in the substrate was maintained at a level of 80% and temperature was maintained in the range of 20–25°C. The model experiment comprised several variants, namely, (1) traditional vermicultivation on PMM for 33 days (VC-1); (2) vermicultivation on PMM after preliminary solid-phase cultivation of the fungus T. viride for 11 days, with the period of vermicultivation per se of 22 days (VC-2); (3) vermicultivation on PMM for 33 days with concurrent introduction of the fungus T. viride into the substrate (VC-3); (4) PMM composting with introduction of the fungus without vermicultivation (PMMTr). During vermicultivation the efficiency of the initial substrate conversion into compost by earthworms was determined in all experimental variants. For this purpose, the fractions of coprolite were separated using 3- and 5-mm sieves to calculate the percent rate of each fraction. In the vegetation experiment, peat supplemented with 25% of PMM was used as a control. The experimental soil variants were composed of the peat supplemented with 25% of the corresponding vermicompost variants (VC-1, VC-2, or VC-3) or 25% of the PMMTr relative to the total weight of the prepared substrate. The experiment was performed in seven replicates using plastic containers (500 ml) with six wheat grains planted in each. The wheat was grown at a temperature of 25 ± 1°C in a phytochamber with daylight of 15 h. Biological activity of vermicompost was assessed according to the increase in wheat green mass (dried to a constant weight) expressed as percent of the control. Growth-stimulating activity was determined by biological testing of wheat seeds soaked for 5 min in an acid extract (pH 4–4.5) of vermicompost. Stimulatory effect was assessed according to the difference between the dry weights of green shoots and roots in the experimental and control (with distilled water) variants expressed as percent of the control. T. viride enrichment culture was produced on wheat grain preliminary boiled to softness in a small amount of water. The grain was placed into bottles; the bottles were stopped and twice sterilized in an autoclave at 1 atm for 30 min with an interval of 1 day. Then, the tubes with the grain inoculated with T. viride were kept in a thermostat at a temperature of 28–30°C until fungal mycelium completely covered the grain. The produced enrichment culture was introduced into the substrate in an amount of 2% of the PMM weight in the container. Since the fungal population on the PGA (potato–glucose agar) medium was reduced due to active growth of the colonies and competition for the substrate, the T. viride during vermicultivation was counted on MPA (meat–peptone agar) [10]. Vermicomposts and initial organic substrate were chemically assayed for the following components: nitrate-nitrogen using phenol disulfonic acid (state standard GOST 26488-85), ammonium nitrogen using the Nessler reagent (GOST 26489-85), labile phosphorus according to Kirsanov (GOST 26207-91), and exchange potassium according to Kirsanov using lame photometry (GOST 26207-91). To assess the impact of the storage period on the vermicomposts quality (growth-stimulating and fungistatic activity) the model experiment with the cultivation of wheat seedlings in peat containing soil was conducted after 3 months of storage. All variants of vermicompost were stored at 5°C. In each experimental variant the soil was supplemented with 25% of previously obtained corresponding vermicompost variants (VC-1, VC-2, VC-3). As a control we used the soil supplemented with 25% of peat-manure mixture (PMM). To evaluate the level of fungistatic activity of vermicomposts the infectious load was artificially modeled in the experiment by adding mycelium and spores of Bipolaris sorokiniana into the soil as a part of the chopped agar medium Capek. The model experiment include the following variants: (1) Soil + VC-1; (2) Soil + VC-1 + Bipolaris sorokiniana; (3) Soil + VC-2; (4) Soil + VC-2 + Bipolaris sorokiniana; (5) Soil + VC-3; (6) Soil + VC-3 + Bipolaris sorokiniana. The experiment was performed in five replicates using plastic containers (500 ml) with six wheat grains planted in each. The wheat was grown at a temperature of 25 ± 1°C in a phytochamber with daylight of 15 h. Data were statistically processed using Statistica v. 5.5 and 6.0 software packages [11]. In the paper, the data are presented as a mean value with a confidenc entervals for variance and standard deviation calculated using Student’s test (p < 0.05). Data on wheat growth in the vegetation experiment were compared according to the nonparametric Mann–Whitney test (p < 0.05). 3. Results and Discussion Determination of the efficiency of organic substrate conversion by earthworms have demonstrated that preliminary fermentation of the substrate with T. viride (VC-2) allowed the yield of vermicompost to be elevated by 8.5% as compared with traditional vermicultivation (VC-1) International Journal of Agriculture and Forestry 2014, 4(5): 343-350 345 as early as day 11 (Table 1). By the end of the experiment, preliminary fermentation (VC-2) provided for 88.7% yield of vermicompost, which is approximately 4.0% higher compared with traditional vermicultivation. Thus, in this case the period of vermicultivation alone providing a high percent yield of vermicompost can be reduced to 11 days. For comparison in variant with traditional vermicultivation (VC-1) it takes at least 22 days. The total period of the organic substrate conversion by T. viride and earthworms takes no more than 22 days (since the periods when fungi and earthworms are used overlap). Decrease in the period of vermicultivation alone as the most energy- and resource-consuming process can decrease the net cost of vermicompost and increase the overall economic efficiency of vermicultivation. Consequently, solid-phase bioconversion of the substrate, which is a modern and promising approach not requiring considerable expenses, makes it possible to more rationally utilize vermicultivation facilities and provide the necessary conditions for both the growth of Trichoderma and bioconversion of the substrate. Such effect was earlier described for wastewater sediments [12]. The results of the vegetation experiment and data of the biological test demonstrate that decrease in the period of vermicultivation did not have a negative effect on the quality of the produced vermicompost, since all the mean values of growth-stimulating characteristics in the variant with preliminary composting of the substrate by T. viride (VC-2) were higher compared with traditional vermicultivation (VC-1) (Table 2). However, differences between the variants were statistically insignificant. Results of the vegetation experiment with wheat demonstrate that the use of vermicomposts VC-2 and VC-3, as well as the PMM inoculated with T. viride (PMMTr), led to a statistically significant increase in wheat green mass relative to the control variant of experiment (without VC or PMMTr). In addition, variants VC-3 and PMMTr demonstrated a statistically significant positive effect as compared with variant VC-1 (by 20 and 31%, respectively) (see Table 2). The most pronounced positive effect in the vegetation experiment was observed in the variant with PMMTr. However, this effect was unobservable in the biological test with PMMTr acid extract and the total content of mineral nitrogen (N-NH4 + N-NO3) in the variant with PMMTr was minimal (Figure 1, Table 3). Presumably, this suggests that the formation of a certain fungal hyphosphere in the root-inhabited layer caused the plant stimulation rather than the substances produced by Trichoderma that accumulated in the substrate. As is known, hyphae during their growth release into the rhizosphere various amino acids, vitamins, organic acids, enzymes, and other compounds, thereby constantly stimulating plant growth [13]. However, this variant of compost in both the agrochemical characteristics and results of biological test displayed a considerably lower efficiency as compared with vermicomposts (see Figure 1 and Table 3). In addition, the T. viride population in the variant of compost without earthworms was higher than in the vermicomposts (see below). Table 1. Yield of vermicompost at different stages of vermicultivation Variants of experiment Traditional vermicultivation on PMM (VC-1) Vermicultivation on PMM after preliminary T. viride cultivation for 11 days (VC-2) Vermicultivation on PMM with concurrent T. viride inoculation (VC-3) Period of cultivation*, days Trichoderma Earthworms — 11 –– 22 — 33 11 –– 22 11 33 22 11 11 22 22 33 33 Yields of vermicompost fractions (coprolite, fraction 3 + 5 mm), % of initial PMM 67.8 84.5 85.0 –– 76.2 88.7 67.2 82.5 87.3 Note: (–) without T. viride inoculation into vermicompost; * For variant VC-2, the total cultivation time is not equal to the sum of fungus and earthworm cultivation times (see Experimental). Table 2. Effect of vermicomposts on the formation of wheat green mass Variants of experiment Wheat green mass, mg Increase in green mass relative to control, % Increase in green mass relative to VC-1, % Number of plants with four leaves, % of total number of leaves Control (peat + PMM) Peat + VC-1 Peat + VC-2 Peat + VC-3 Peat + PMMTr 33.28 ± 1.62 36.54 ± 2.50 40.81 ± 1.98 43.87 ± 1.91 47.92 ± 3.55 –– 9.8 22.62 31.82 44.02 –– –– 11.68 20.06 31.14 6.06 9.75 12.50 30.95 54.76 346 Natalia N. Tereshchenko et al.: Using Trichoderma viride for Optimization of Vermicomposting Processes to Improve the Quality of Vermicompost and Prolong the Storage Period Table 3. Content of nutrient elements in the composts produced in different variants of experiment (see Experimental), mg/kg of absolutely dry substrate Variants of experiment Initial PMM VC-1 VC-2 VC-3 PMMTr N-NH4 262 137 91.5 194 212 N-NO3 120 869 1554 1170 470 K2O 8556 9954 13447 8671 10620 P2O5 4850 6120 8027 6558 6511 Wheat plants grown on vermicomposts modified with T. viride (VC-2 and VC-3) exceeded, in dry weight, the plants grown on traditional vermicompost (VC-1) (see Table 2). In addition, the stimulatory effect of Trichoderma in these variants appeared in the maximal number of plants with four completely developed leaves as compared with the control and VC-1 (see Table 2). Comparison of three vermicompost variants has demonstrated the most pronounced positive effect of VC-3, produced by concurrent inoculation with T. viride and earthworm cultivation (see Table 2). The stimulatory effect of Trichoderma, observed in the experiment, agrees with the data of several researchers [1, 14–16]. According to the data of the biological test shown in Fig. 1, treatment with acid extracts prepared from vermicomposts and PMMTr provided for a statistically significant increase in the wheat green mass and root weight relative to the control. However, the differences between the experimental variants were statistically insignificant except for the wheat green mass in variants VC-3 and PMMTr (see Figure 1a). Figure 1. Efficiency of acid extracts derived from the vermicompost variants (VC-1, VC-2, VC-3) and compost produced by T. viride fermentation (PMMTr) on the root weight (a) and green mass (b) of wheat seedlings Figure 2. Dynamics of T. viride population in the vermicompost variants (VC-1, VC-2, VC-3) and compost produced by fermentation of organic substrate with T. viride (PMMTr) International Journal of Agriculture and Forestry 2014, 4(5): 343-350 347 Despite the fact that statistically significant differences between the vermicompost variants in vegetation experiment and bioassay were not obtained the increase in green mass of wheat seedlings in the sequence VC-1

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