Development of Forestry Species Inoculated with Trichoderma spp . Fertilized with Rock Phosphate

The study aimed to determine the effects of inoculation of Trichoderma spp. on the development and extraction of nutrients from Eugenia pyriformis Cambess. (uvaieira) and Myrcianthes punges (O.Berg.) (guabiju) subjected to fertilization with rock phosphate. The treatments used at planting were: Control; Trichonat PM; Trichoderma sp. FS1; Rock phosphate; Trichonat PM + Rock phosphate; and Trichoderma sp. FS1 + Rock Phosphate. The following characteristics were evaluated at 540 days after planting: plant height, crown diameter, branch insertion points, biomass, and nutrient content. The uvaieiras inoculated with Trichonat PM and Thrichoderma spp. showed higher vegetative growth and higher values of N, P, K, Ca, Mg and S, respectively. For guabijuzeiros, the treatment with Trichoderma spp. presented the highest insertion point of the branches without phosphorus fertilization However, the treatments did not affect nutrient extraction. Thus, Trichoderma spp. inoculants have the potential to promote vegetative growth and nutrient extraction in plants.


INTRODUCTION
Forest species have a great source of vitamins, antioxidants, and essential oils.Thus, research in these areas has increased to meet the demand from the food, cosmetics and pharmaceutical industries (Franzon et al., 2010;Marin et al., 2008).Management of fruit plants in preservation areas or on small properties can become a practice that combines environmental conservation and generates income to small farmers.Myrtaceae is a species that can be used for restoring vegetation (Lorenzi, 2002;Pirola, 2013).
The Myrcianthes punges (O.Berg.)(popularly known in Brazil as guabijuzeiro) and the Eugenia pyriformis Cambess.(popularly known in Brazil as uvaieira) which are native trees to the Southern Region of Brazil are among the plants belonging to the Myrtaceae Family.Myrcianthes punges is a perennial forest fruit tree with slow growth and Eugenia pyriformis is also a perennial forest fruit tree, but with fast growth and early fructification.Both can be used in forest management systems for reforestation of degraded areas and ciliary recomposition (Carvalho, 2010).
Technical and scientific information regarding nutritional requirements and their reflection on the productivity and quality of uvaia and guabiju fruits are scarce.The phosphorus nutrient is of paramount importance for fruit species in general, as its deficiency is marked by a delay in plant and root growth, a delay in flowering, inhibition of lateral bud sprouting and reduced quantity of fruits; and consequently, a reduction in productivity (Araújo & Machado, 2006).
According to Lima et al. (2011), phosphorus favors roots and seedlings to develop faster, increases resistance to cold, improves water use efficiency, promotes disease resistance in some plants and increases nutrient absorption.The use of rock phosphate in its natural form is very restricted due to its low solubility, which requires an association of this material with other sources of soluble phosphorus.To increase phosphorus solubilization in natural sources, studies have been conducted with the inoculation of phosphorus solubilizing microorganisms (Novais et al., 2007).
In this context, the use of the Trichoderma spp.Fungus is highlighted.It is a natural inhabitant of soils and highly interactive in the root and in the interior of the plants through different action mechanisms such as competition, parasitism, and antibiosis against microorganisms capable of causing disease in plants (Bedendo et al., 2011).This fungus aids in decomposing and mineralizing plant residues, as well as promoting P solubilization that contributes to the availability of this nutrient to the plants, allowing greater and faster absorption (Jesus et al., 2011).With the objective of evaluating the in vitro phosphate solubilization potential, Oliveira et al. (2012) used seven isolates of Trichoderma spp.and found that all were able to solubilize calcium phosphate in the culture medium.
In addition to helping in the solubility of nutrients necessary to plants, the Trichoderma genus can promote their growth due the production of Indole-3-acetic acid (IAA) (Hoyos-Carvajal et al., 2009;Carvalho et al., 2008).Fortes et al. (2007) found that the Trichoderma isolate (E15) promoted eucalyptus micro-cut rooting (Eucalyptus sp.), showing a significant increase in the rooting percentage.
In this sense, the objective of the study was to evaluate the effect of inoculating Trichoderma spp.fungus and fertilization with rock phosphate on the initial development and nutrient extraction of Eugenia pyriformis and Myrcianthes punges plants in field conditions.

Obtaining seedlings
Myrcianthes punges and Eugenia pyriformis seeds were obtained from a forest fragment in the municipality of São José das Palmeiras in the western region of the State of Paraná, Brazil, and seeded in plastic pots measuring 45 x 17 cm and 14 cm in height (two seeds per pot), with MECPLANTA FSC  as substrate registered at MAP PR 0954910001-0 as an "F" class soil conditioner (composition of the product: pine bark, vermiculite, corrective acidity, mineral fertilizers) and ravine soil at the ratio of 1:1, and then kept in a greenhouse under intermittent mist.The seedlings were transferred to polyethylene bags (10 x 20 cm) after 60 days with a capacity of 500 ml of MECPLANTA FSC  substrate and then remained in the greenhouse for another 90 days.The seedlings were planted in the second half of September 2011, into previously prepared pits with 2x2 m spacing and dimensions of 40 x 40 x 40 cm.

Implemented products
The commercial product Trichonat PM  (Natural Rural S.A., Araraquara-SP) was used as an inoculum source of Trichoderma spp.fungi, composed of a powder formulation at a concentration of 6x10 6 conidia g -1 .A sample of Ficus carica L. rhizospheric soil containing Trichoderma was transferred to Petri dishes containing potato-dextrose-agar (PDA) culture medium to obtain Trichoderma spp.FS1 isolates, and the plates were subsequently incubated in a growth chamber (BOD = bio-oxygen demand) at 25 °C with a photoperiod of 12 h for 7 days.After this period, 5 mm diameter discs of PDA culture medium containing Trichoderma mycelium were added to glass vials containing 5g of sterile parboiled rice, and the vials were placed in a BOD chamber/incubator at 25 °C with a photoperiod of 12 h for 20 days.
The rock phosphate used was Arad origin, which generally has 10 to 12% of P 2 O 5 soluble in citric acid and total P 2 O 5 contents between 28 and 30%.

Field experiment
The experiment was carried out in Guarapuava, south-central state of Paraná, Brazil, with geographic coordinates of 25° 23' 36" S, 51° 27' 19" W and 1,120 m altitude.According to Köppen, the region's climate is classified as humid subtropical Cfb with mild summers, frosty winters, and annual rainfall of 1,800-2,000 mm (data provided by the Technological Institute of Meteorological System of Paraná).

Evaluations
The following characteristics were evaluated at 540 days post planting in July 2013: a) plant height: a measuring tape (cm) was used from the base of the plant stem until the end of the crown; b) canopy diameter: a measuring tape on two equidistant sides (cm) was used, c) insertion point of the branches: a measuring tape from the base of the plant stem to the insertion point of the first branch (cm) was used.
After the measurements, the plants were completely removed from the soil for chemical analysis.The plants were excavated and divided into roots, stems, branches, and leaves.The samples were dried at 70 °C in a forced-circulation oven with air renewal.Fresh and dry mass for all parts were evaluated with a precision digital scale.Then, the samples were ground into fine particles in an electric knife mill with a 2 mm sieve, and stored in a cool place at room temperature until the chemical analyses were carried out.
The macronutrients contents from the shoot and the root of the plants were determined according to the methodology described by Malavolta et al. (1997).
The N concentrations in the dry tissues were determined by digestion using sulfuric acid and semi-micro Kjeldahl method.After the nitric-perchloric acid digestion, phosphorus (P) was determined by molecular absorption spectrophotometry, sulfur (S) by barium sulphate turbidimetry, potassium (K) by flame photometry and calcium (Ca) and magnesium (Mg) nutrients by atomic absorption spectrophotometry.
The nutrient content was calculated according to the dry mass and the nutrient content in the plant (= dry mass x nutrient content) (Malavolta et al., 1997).
The results were submitted to analysis of variance and the means were compared by the Tukey test when significant at 5% probability level using the statistical program SISVAR 5.0 (Ferreira, 2011).

Vegetative growth
Regarding the evaluations of the Eugenia pyriformis species, there was no interaction for the variables plant height and crown diameter between the factors.However, a significant effect was observed for the inoculum factor, regardless of the phosphate fertilization.The Trichonat PM  treatment had the highest plant height and crown diameter when compared to the control, which was not statistically different from the treatment with Trichoderma spp.FS1 (Figures 1A and 1B).
There was an interaction between the factors observed for the biomass and first branch insertion point evaluations in Eugenia pyriformis (Figures 1C and D).The treatment with Trichoderma spp.provided the highest biomass, followed by the Trichonat PM  treatment, which differed from the control treatment in the absence and presence of phosphate fertilization.However, only the Trichonat PM  inoculum presented a statistical difference in the phosphate fertilization, increasing plant biomass by 36%, when compared to the absence of phosphate fertilization (Figure 1C).On the other hand, the Trichoderma spp.FS1 inoculum significantly increased the insertion point of the first branches of Eugenia pyriformis in the presence of phosphate fertilization, and it was statistically different from the control treatment (Figure 1D).

Guarapuava-PR, 2013.
Regarding the vegetative growth assessments of Myrcianthes punges, no significant differences were found for the variables crown diameter, plant height and biomass.For the insertion point of the first branches variable, there was an interaction between the inoculum and fertilization factors.The Trichoderma spp.treatment increased the insertion point of the branches by 130% when compared to the control in the absence of fertilization, differing statistically from those with the presence of phosphate fertilization (Figure 2).Trichoderma spp.inocula, Guarapuava-PR, 2013.

Nutrient extraction
A significant interaction was found between the factors of nutrient extraction results in Eugenia pyriformis plants (Table 1).In plants fertilized with rock phosphate, the Trichoderma spp.FS1 treatment provided the highest content of N, P, K, Ca and Mg in the plants increasing by 160, 150, 133, 210 and 133%, respectively, in relation to the control without inoculation.
However, the commercial Trichonat PM  inoculum in the absence of phosphate fertilization had higher values for most of the macronutrients, followed by   Trichoderma spp.FS1, which differed significantly from the control treatment.For the P, K, and Mg elements, the increases were 300, 271 and 150%, respectively, in relation to the control.
For nitrogen, the Trichoderma spp.FS1 inoculum had higher extraction.For the sulfur macronutrient, an effect was only observed for the inoculum factor, in which the maximum content was observed in the Trichonat PM  treatment (1.11 g of S plant -1 ), which did not significantly differ from the Trichoderma spp.FS1 treatment (0.67 g of S plant -1 ).

DISCUSSION
Both species showed greater vegetative development when using Trichoderma spp. as verified by increased plant height, canopy diameter, biomass or the insertion point height of the branches.Similarly, Maciel et al. (2012) also found that Eucalyptus saligna seedlings showed higher height when planted in substrate with the commercial product Trichodel  [composed of selected strains of the Trichoderma spp.fungus (1x10 9 of viable cells per mL)].In sour passion fruit (Passiflora edulis var.flavicarpa Degener), effects of Trichoderma sp.inoculation (native TCN-014) have been also verified, in which larger stem length, stem diameter, true leaves, root length and total dry weight were observed two months after seedling establishment compared to TCC-005, the commercial strain of Trichoderma harzianum (Cubillos-Hinojosa et al., 2009).
These results indicate that the Trichoderma spp.FS1 and the Trichonat PM  directly promote plant growth through the production of hormones (auxins, cytokinins, and ethylene) or by supplying the nutrient requirements for solubilization (Machado et al., 2011;Oliveira et al., 2012).The production of Indole-3-acetic acid in eucalyptus mini-cuttings treated with Trichoderma CEN 262 isolate increased on 136%, 136% and 43% of the shoot dry mass, root dry mass and mean height of the plants, respectively, compared to untreated plant cuttings (Carvalho et al., 2008), In this study, Eugenia pyriformis plants notably showed higher nutrient extraction when inoculated with Trichoderma spp.for the elements nitrogen, phosphorus, potassium and magnesium.Similarly, Machado et al. (2011) found an increase in the mineral nitrogen absorbed by Avena strigosa Schreb (black oat) in plants inoculated with Trichoderma harzianum and nitrogen fertilization.In another study, the phosphorus content in coffee leaves was higher when submitted to Organic CS  organic soil conditioner (composed of millet colonized by the Trichoderma asperellum fungus at a concentration of 1x10 8 CFU/g) (Jesus et al., 2011).
According to the results, a difference in the efficiency of Trichoderma sp.FS1 and Trichonat PM  was observed, which according to Ethur (2006), may be related to the ability of each isolate to produce secondary metabolites and to compete with other organisms in the rhizosphere environment of the plant.
The results show the potential of using Trichoderma sp.FS1 isolates and Trichonat PM  to promote vegetative growth in Eugenia pyriformis regardless of phosphate fertilization.Isolates of Trichoderma sp.FS1 increased biomass and nutrient content of plants in the presence of phosphate fertilizers, while Trichonat PM  provided higher nutrient extraction in non-fertilized plants.
Myrcianthes punges is a slow-growing fruit tree, and it has not been clearly benefited by localized fertilization with rock phosphate or by the inoculation of Trichoderma spp. in the first years of development.Moreover, different types of management may favor or inhibit the establishment of microbial groups in the rhizosphere (Cardoso et al., 1992).Similar results were observed by Macedo & Teixeira (2012)  However, it is important to continue studies for a longer period of time to better elucidate the results in Myrcianthes punges.

CONCLUSION
The best results in growth and nutrient extraction were found when using Trichonat PM  without fertilization, and Trichoderma sp.FS1 with phosphate fertilization in Eugenia pyriformis plants.
FS1 promoted a greater insertion point of the first branches However, the treatments did not influence nutrient extraction by the plants.

Figure 1 .
Figure 1.Plant height (A), crown diameter (B), biomass (C) and the insertion point of the first branches (D) Eugenia pyriformis subjected to several Trichoderma spp.Inocula.

Figure 2 .
Figure 2. Insertion point of the first branch of Myrcianthes punges subjected to several.
when evaluating the effect of liming and phosphate fertilization on the growth of Eugenia stipitata McVaugh seedlings (popularly known as araçá-boi), finding that high doses of phosphorus in the substrate affected the nutrient availability, hindering the vegetative development of this plant.Based on the results, Tichoderma spp.(a natural and highly interactive soil resident in the root and interior of plants) promoted vegetative growth and increased nutrient content in Eugenia pyriformis.

Table 1 .
Effect of different Trichoderma spp.inocula with or without phosphorus fertilization (phf) on the extraction of Eugenia pyriformis nutrients.

plant -1 ) Mean Without phf With phf
*Means followed by the same lowercase letter in the column and the same capital letter on the line do not differ from one another according to the Tukey test (p < 0.05).phf = phosphate fertilization (1 kg plant -1 of ARAD rock phosphate); ns = Not significant.

Table 2 .
Effect of different Trichoderma spp.inocula with or without phosphorus fertilization (phf) on the extraction of Myrcianthes punges nutrients.