Biostimulant and Nematicidal Effect of Trichoderma harzianum Rifai and Aqueous Extract of Azadirachta indica A. Juss. in Solanum lycopersicum L

The experiment was carried out with the objective of evaluating the biostimulating and nematicidal effect of Trichoderma harzianum Rifai "strain A-34" and aqueous extract of Azadirachta indica A. Juss. (neem) in Solanum lycopersicum L. (tomato) cv. "PR-92". Two trials were developed, the first in the seed germination process and the second in potting conditions for the treatments interaction and the inoculation of Meloidogyne spp. with tomato seedlings. The results showed that tomato seeds cv. “PR-92” treated with T. harzianum "A-34" and aqueous extract of neem leaves did not affect the percentages of germination, however, the combined use of the preparations promote the seedlings growth. It was also corroborated that the seedlings infested with Meloiodgyne spp. showed lower means for stem length, total fresh mass and dry mass variables, compared to non-infested ones. As for the regulation of Meloidogyne spp., a reduction of the gill index was achieved in 53.5%, 38.5% and 61.5% for treatments where T. harzianum, neem and their combination were respectively applied with significant differences respect to control.


Introduction
The production and fruit quality in tomato crops depends on an adequate control of fungi, phytopathogenic bacteria and insects that damage foliage and fruits, also the root-knot nematodes and weeds that compete with plants for moisture and nutrients [1]. Phytopathogenic nematodes are at the forefront of pests affecting this crop in the world, especially galls formers (Meloidogyne spp.). Moosavi [2] states that cause 12.3% losses in agriculture in the world, and around 5% of losses are attributed to Meloidogyne spp.; considered as important pests of many cultivated plants [3]. The root nematode (Meloidogyne incognita) is one of the major limiting factors affecting growth and yield, causing an estimated $ 100 billion loss per year worldwide [4].
At present, the use of biological mediums and botanical preparations are booming as non-chemical alternatives in the management of Meloidogyne spp., because they reduce the economic-environmental impact and frequently promote the plants development [5].
One of the biological alternatives for the Meloidogyne spp. management is the use of antagonist fungi of the genus Trichoderma. Several species of Trichoderma have been evaluated as biological control agents against nematodes in various crops and experimental conditions, emphasizing among the mechanisms involved the parasitism, enzymatic lysis, antibiosis and induced resistance [6]. The increase of plant growth, yield and other parameters could be attributed to the release of growth promoting substances by Trichoderma spp. and other biological agents studied in the control of M. incognita [7].
The use of plants with properties or characteristics that promote the regulation of populations of Meloidogyne spp., constitutes a very promissory practice for the management of this pest, especially for the development reached in Cuba urban agriculture. An example of a plant with nematicidal activity is Azadirachta indica A. Juss., plant belonging to the family Meliaceae, with biological activities widely used in agriculture for the management of insect pests [8] and nematodes [9]. Several authors reported reduction in gill index by Meloidogyne spp. through biodesinfection with foliage of A. indica [10] and different doses of A. indica and Aqueous Extract of Azadirachta indica A. Juss. in Solanum lycopersicum L.
wastes [11], although they recommend to deepen the effect over plant growth.
Taking into account the above, the research was carried out with the objective of evaluating the biostimulating and nematicidal effect of T. harzianum "strain A-34" and aqueous extract of A. indica in S. lycopersicum.

Materials and Methods
The research was carried out at the Microbiology Laboratory and Experimental Areas of the Forestry and Agriculture Sciences Faculty in University of Pinar del Río, Cuba, located at 22° 24'48''N and 83° 41'16' 'W, between September and December 2014.

Description of the Experiment I
The effect of T. harzianum "strain A-34" and aqueous extract of A. indica on germination of tomato seeds was evaluated, considering the treatments described in table 1. The plant material used was S. lycopersicum cv. "PR-92". After treatment, the seeds were placed on filter paper for drying in laboratory conditions at 25.6°C temperature. Six hours later they were randomly distributed into Petri dishes of 10 cm diameter, using five plates per treatment and 50 seeds per plate. They were subjected to a dark chamber for 12 hours and subsequently to a light and dark regime at the same period of time up to 94 hours after sowing, with evaluations at six hour intervals.
Germination was quantified at six-hour intervals and the germination percent (GP), germination rate index (GRI) [12] and mean germination time (MGT) [13] were evaluated. Calculations were made by the formulas described below.

Description of the Experiment II.
The experiment was developed in climatic conditions characterized by temperatures between 18.2 and 28.9 °C, with an average of 22.6 °C, and average relative humidity of 79%. The plant material used was S. lycopersicum cv. "PR-92". Four treatments were established in S. lycopersicum plants with and without inoculation of Meloidogyne spp., following a completely randomized design with factorial arrangement ( Table 2). Table 2. Treatments evaluated in experiment II.

Number
Treatments Tag Twenty pots with a capacity of 900 g of substrate were used in each treatment, 10 with inoculation of nematodes (Meloidogyne spp.) in the rhizosphere and the remaining ones without inoculation of nematodes. As a substrate, a mixture of Ferralitic Yellowish Leached Soil [14], peat and rice husk, was used to obtain a ratio of 70: 20: 10, respectively. In soil disinfection, 4% formaldehyde was used for 72 hours, whereas peat and rice husk were sterilized at a temperature of 121°C in an oven during four hours.
Sowing was done manually, placing four seeds per bag, leaving two seedlings per bag after germination with uniformity in the choice of the same.
The evaluations were carried out 35 days after germination, at which time 10 seedlings were randomly extracted from each treatment and processed in the Laboratory of Microbiology at Pinar del Río University. It was determination the galls index [15], shoot length and diameter, leaf number, total fresh mass and total dry mass, foliar and root mass.

Obtaining, Preparation and Application of
Trichoderma and Neem.
The T. harzianum "strain A-34" bioprepared was obtained at the Reproduction of Entomophages Center of "La Conchita", in Pinar del Río municipality. It had a concentration of 1.9 x 10 9 cfu ml -1 , 100% purity and 96% viability. The applications were carried out three days before planting and two others every seven days, using a dose of 10 ml/pot of conidial solution, obtained at a concentration of 2.5% (w/v) of the solid bioprepared with distilled water.
In the preparation of the aqueous extract was used neem leaf collected from trees located at 22° 25'11 "N 83° 41'17" W, in organoponics of the municipality of Pinar del Río. The foliage was subjected to a drying process under ambient conditions, in the shadow, until losing between 90 and 95% Universal Journal of Agricultural Research 5(5): 251-256, 2017 253 humidity. The preparation was carried out in a glass vessel, with a proportion of 50 g l -1 of crushed foliage in distilled water during 8 hours, later the extract obtained was filtered. Three applications were carried out at a rate of 15 ml / pot from five days after germination with a seven day interval.

Preparation and Application of the Inoculum of
Meloidogyne spp.
Samples of Ferralitic Yellowish Leached Soil [14] were collected from the experimental area of the Pinar del Río University, where infestation by Meloidogyne spp. in S. lycopersicum was detected. The population of Meloidogyne spp. was extracted through bioassay method by indicator plant, using Cucurbita maxima. The analysis of the species composition showed the presence of M. incognica and Meloidogyne sp. to a less extent. The application of the inoculum was carried out in the rhizosphere five days after germination, guaranteeing 0.5 J 2 -eggs per gram of soil in each pot.

Statistical Analysis Employed
A simple and multivariate analysis of variance was performed using the Duncan Multiple Rank test for mean comparison, with a confidence level of 95% (p≤0.05). Linear regression analysis was also applied for germination dynamics. The statistical program SPSS version 21.0 for Windows was used.

Results
In experiment I, germination was initiated 30 hours after sowing for all treatments, with a linear increase up to 66 hours expressed in coefficients of determination (R 2 ) higher than 97%, although the germination percent increased significantly at 48, 54 and 60 hours with T. harzianum "strain A-34", whereas it was reduced in seeds treated with neem aqueous extract. The germination percent at 66 hours was 91.7%, 86.5% and 84.5% in treatments T, C and N, respectively. Table 3 shows similar values of germination rate index (GRI) in treatments T, N and C (1.5, 1.29 and 1.41), although the mean germination time (MGT) was affected in the treatment N (51.13), with significant differences regarding T and C (47.2 and 48.8). In addition, similar results were achieved in percentages of germination at 96 hours, with values above 96%.   In experiment II the galls index was reduced by Meloidogyne spp. in 53.5%, 38.5% and 61.5% for T, N and T+N treatment, respectively, with significant differences over control (figure 2), where a galls index of 1.6 grade was determined.
The analysis of variance for the effect of the evaluated factors and their interaction on the growth variables of S. lycopersicum cv. "PR-92" is presented in table 4. Except for shoot diameter, leaf number and leaf dry mass, the other variables showed significant differences between infested plants and those not infested with Meloidogyne spp. For the treatment factor, there was a significant effect over all variables evaluated. The interaction was only significant for shoot length, indicating strong influence of the factors evaluated on this variable.  Table 5 shows the average values of the growth variables in S. lycopersicum cv. "PR-92" with and without nematode inoculation (Meloidogyne spp.). It should be noted that all variables reached lower means in infested plants (with nematodes) than in non-infested plants (without nematodes), although the greatest reduction in growth was expressed in shoot length (10.4%), total fresh weight (12.9%), dry weight (17.3%), dry leaf weight (14.2%) and dry weight (36.4%).  Table 6 shows the effect of treatments on the growth of S. lycopersicum cv. "PR-92". Higher means could be observed in seedlings treated with Trichoderma (T) and their combination with neem (T + N), which showed significant differences in reference to those treated with neem (N) and untreated (C). Application of aqueous neem extract did not affect the growth of S. lycopersicum cv. "PR-92", since it shows means similar to the control in the evaluated variables.

Discussion
Seed treatment of S. lycopersicum cv. "PR-92" with T. harzianum "strain A-34" and aqueous extract of 5% (w/v) neem leaves has caused a linear growth in the germination dynamics, without affecting the germination percentage, even though between 42 and 60 hours after sowing significantly lower means were manifested when the seeds were treated with neem leaf aqueous extract. Germination rate and mean germination time indexes were also verified with little variability, although in the last one it was observed a reduction in seeds treated with Trichoderma and an increase of 2.35 hours with aqueous extract of neem leaf. The use of T. harzianum "A-34 strain" promote the germination, a result that could be related to the reports on the production of growth factors (auxins, gibberellins and cytokinins) by T. harzianum, which are released into the medium and stimulate germination and plant development [16][17], whereas the results with the aqueous extract of neem leaf suggest that the uniformity of germination is not affected. The effect of T. harzianum during 15 days after inoculation on passion fruit seeds (Passiflora adulos Degener), showed significant increases in germination after four days after inoculation [18]. Recent study on the effect of T. harzianum "strain A-34" inoculated in seeds of S. lycopersicum cv. "Vyta", reports absolute germination percent higher than control [19].
The use of T. harzianum "strain A-34" and aqueous extract of neem leaves evidenced a reduction in the agglutination index by Meloidogyne spp., although the infestation provoked with the level of inoculum used is considered low, however it is be observed that agglutination index ≤ to grade 2 caused significant reductions in shoot length, total fresh mass and total and radical dry mass of S. lycopersicum cv. "PR-92", which corroborates the phytoparasite action of Meloidogyne spp., Although it would be opportune to evaluate different levels of inoculum.
It was also possible to observe higher growth of S. lycopersicum cv. "PR-92" in seedlings treated with Trichoderma and its combination with aqueous extract of neem, an aspect that is favorable in the search of alternatives that can be integrated in the management of Meloidogyne spp. and that at the same time can promote, or not affect, the growth of plants. The results corroborate the biostimulating effect of Trichoderma and suggest that aqueous extract concentrations of neem ≤ 5% do not affect the growth of S. lycopersicum cv. "PR-92".
In the treatments where T. harzianum was used, it was reduced the number of galls compared with the control [20]. Several authors report a significant reduction of the agglutination index by Meloidogyne spp. with different forms of use of neem preparations in S. lycopersicum [2,11,21]. Applications of neem cake extract and T. harzianum caused a reduction in the number of galls and egg masses in 70.3% and 59.3%, respectively [22].
Trichoderma species may induce changes in plant morphology and development, with increasing root and/or foliar biomass being the most common expression of growth promotion [23]. In treatments where T. harzianum was applied, increases were reported in relation to the control for the total fresh and dry mass of the plants [7]. There was also an increase in shoot length with different forms of use of neem preparations in tomato plants inoculated with M. javanica [21], although there were no significant differences for this variable in S. lycopersicum cv. "Campbell 28" when used in biodesinfection of soil infested with Meloidogyne spp. [11].

Conclusions
The results evidenced biostimulating effect of T. harzianum "strain A-34" on the germination and growth of S. lycopersicum cv. PR-92, whereas the aqueous extract of 5% neem leaf does not limit these processes, although it causes a delay in the seed germination without affecting the germination percentage. A regulatory effect on Meloidogyne spp. with the use of Trichoderma, neem and their combination suggests deepening the use of these biopreparates as alternatives that offer different benefits when used in the management of plant parasitic nematodes.