Growth promotion and productivity of lettuce using Trichoderma spp . commercial strains

The aim of this study was to evaluate four strains of Trichoderma spp. (T. harzianum IBLF 006 WP, T. harzianum IBLF 006 SC, T. harzianum ESALQ 1306 and T. asperellum URM 5911) for seedling growth promotion in laboratory and head lettuce yield in field conditions. The experiment was carried out in a completely randomized design with four treatments (strains): IBLF 006 WP, IBLF 006 SC, ESALQ 1306 and URM 5911 and a non-inoculated (without Trichoderma) control. Each treatment consisted of 200 seeds, arranged in four replicates. Lettuce seeds cv. Astra were treated with 2 mL Trichoderma suspension (2.5 x 108 conidia mL-1 per each 100 g seeds) and submitted to growth assay in laboratory up to 7 days after sowing. For field experiment, we opened furrows, which were manually sprayed with 5 x 107 conidia mL-1. Afterwards, seedlings were transplanted (4 to 6 leaves of head lettuce cv. Mauren) and harvested 40 days later. Each treatment consisted of four replicates (1.2 x 1.2 m, 16 plants per plot) arranged in randomized blocks. In both experiments, a control without Trichoderma application was included, and we evaluated shoot length, root and total length, shoot, root and total fresh mass and shoot, root and total dry mass, shoot mass ratio, root mass ratio and shoot/root ratio. The germination (%) was evaluated by laboratory tests, whereas in field experiment, height, stem diameter, head diameter, number of leaves and yield were evaluated. The T. harzianum strain ESALQ 1306 provided the best head lettuce growth rate in laboratory test, which was confirmed in field experiment, in which the productivity (50.2 t ha-1) was superior when compared to the other strains (41.38 to 44.23 t ha-1) and the control (30.18 t ha-1).

Received on January 25, 2018; accepted on January 24, 2019 concerning product quality.That's why, lettuce consumption tends to increase and, consequently, the need to produce it in quantity and with superior quality (Barros Júnior et al., 2010).
Trichoderma fungus is considered a free-living soil microorganism, which survives in tropical and temperate regions, being three species (T.harzianum, T. virens and T. viride) the most used in biological control of diseases and on growth promotion of several crops (Hoffmann et al., 2015).Many studies point out the use of Trichoderma to promote initial growth of many commercial crops, such as tomatoes and beans (Chacón et al., 2007;Guimarães et al., 2014), as well as an increase in grain yield and leaf crops (Azevedo Filho et al., 2011;Carvalho et al., 2015a).
The most common method of application of Trichoderma is seed treatment.However, for some crops which are initially installed in seedbeds, when aiming to colonize soil by certain strains of Trichoderma, treatment of substrate or cultivation soil per application via furrow or superficial is an efficient method (Carvalho et al., 2015b).Some isolates of Trichoderma provide plant growth stimulus, producing antibiotics, enzymes and metabolites, whose activities are comparable to plant hormones (Carvalho et al., 2011a).Several strains are excellent producers of secondary metabolites, being volatile or non-volatile and supposed biosynthesizers of nonribosomal peptides, terpenoids, pironas and polyketides and auxin analogs (Machado et al., 2012).Trichoderma spp.insertion into soils poor in mineral nutrients increases the solubilization of these nutrients.The same act as root growth biostimulant, increasing the assimilation of essential nutrients for the plant and contributing to obtain greater productivity, besides promoting root system protection against soil pathogens (Benítez et al., 2004).Such insertion is highly desirable because, in addition to the described benefits, it is possible to reduce or abolish the use of highly soluble chemical fertilizers, which for sustainable agricultural production cause damages to the environment (Azarmi et al., 2011).
Many studies on using live strains for the treatment of seeds and substrates for initial growth promotion of several crops or even in disease control can be found in literature.However, knowledge gaps concerning potential productivity of lettuce with live strains of Trichoderma spp.can be verified.The aim of this study was to evaluate four commercial products based on Trichoderma spp. in seedling growth promotion and head lettuce productivity in field.

Evaluated commercial strains
The commercial strains evaluated in this study were Head lettuce seeds, cv.Astra, were treated with 2 mL of Trichoderma suspension (2.5 x 10 8 conidia mL -1 for each 100 g seeds).Afterwards, the seeds were uniformly distributed on blotting paper sheets, contained in gerbox-type transparent acrylic boxes (11 x 11 cm).Then, these seeds were kept in a BOD type seed germinator (Fanem 347 ® ), at 25ºC, for seven days.We used completely randomized experimental design, with four treatments (strains): IBLF 006 WP, IBLF 006 SC, ESALQ 1306 and URM 5911.One treatment, without Trichoderma inoculation, was included as control.In each treatment, 200 seeds were divided into four replicates (Gerbox) of 50 seeds (Carvalho et al., 2011b).
Germination (%) was obtained by evaluating normal seedlings (absence of necrosis and pathogen in seedlings, seminal and secondary roots without deformation and discounting the dead seeds), shoot length (SL), root length (RL), total length (TL = SL + RL ), shoot fresh mass (SFM), root fresh mass (RFM), total fresh mass (TFM = SFM + RFM), shoot dry mass (SDM), root dry mass (RDM), total dry mass (BIO = SDM + RDM), shoot mass ratio (SMR = SDM/BIO), root mass ratio (RMR = RDM/BIO) and shoot/root ratio (S/R = SDM/RDM).In order to obtain SDM and RDM, shoots and roots were taken out and dried in an oven at 65 o C until constant mass in order to obtain values in milligrams.
Productivity of lettuce plants and their components in field using planting furrow treatment with suspension of Trichoderma spp.
The experiment was installed in a 40.8 m 2 area (34.0 x 1.2 m); soil was plowed and harrowed.Afterwards, seedbeds were prepared with a plow.Chemical fertilizations, as well as soil correction, were done according to Yuri et al. (2002).Then, furrows were opened and 40 mL of Trichoderma spp.suspension (5 x 10 7 conidia mL -1 ) was applied per linear meter using a manual sprayer (550 mL), totalizing 2 x 10 conidia per linear meter in the furrow (Carvalho et al., 2011a).
Right after spray application, head lettuce seedlings, cv.Mauren with 4leaves, grown in 200-cell styrofoam trays, filled with inert substrate, based on peat, carbonized rice chaff, vermiculite and ashes, were manually transplanted (4 plants per 1.2 linear meter).The experimental design was of randomized blocks with four treatments (strains): IBLF 006 WP, IBLF 006 SC, ESALQ 1306 and URM 5911 and one control without Trichoderma spp.inoculation.Each treatment was composed of four experimental plots with four planting lines, 1.2x1.2m total area with 16 plants spaced 0.3x0.3m, plus 0.5 m of a seedbed without lettuce planting for separation between plots.
Forty days after seedling transplant, four central plants of each plot (useful area) were manually harvested to measure height (H), stem diameter (SD), head diameter (HD), number of leaves (NL), shoot fresh mass (SFM), root fresh mass (RFM), total fresh mass (TFM), shoot dry mass (SDM), root dry mass (RDM), total biomass (BIO), shoot mass ratio (SMR), root mass ratio (RMR), shoot/root ratio (S/R) and productivity, using a caliper, a precision scale and a graduated scale.One control treatment was included in order to compare the treatments.Weed control was manually performed.The authors did not verify any pests or diseases.

Statistical analysis
Experimental data were submitted to variance analysis (ANOVA) and to Scott-Knott test (P≤0.05),using computer statistical software SISVAR 5.3 (FERREIRA, 2011).

RESULTS AND DISCUSSION
In relation to germination percentage (GP), T. harzianum strain ESALQ 1306 and T. harzianum IBLF 006 SC were superior to the other treatments, providing 93.50% and 93% of PG.Followed by treatments T. harzianum IBLF 006 WP and T. asperellum URM 5911 and the control, which showed the lowest PG (82%) (Table 1).T. harzianum strain ESALQ 1306 showed a superior increase when compared with the other treatments in relation to shoot length (SL= 2.46 cm), root length (RL= 4.96 cm) and total length (TL= 7.41 cm).For the other strains values of SL, RL and TL ranged from 1.76 to 1.83 cm, 3.78 to 4.27 cm, 5.62 to 6.03 cm, respectively.Control showed the lowest values of SL, RL and TL: 1.54, 3.27 and 4.82 cm, respectively.
For total fresh and total dry mass, T. harzianum strain ESALQ 1306, again, showed to be superior to the other treatments, considering TFM of 23.62 mg and BIO of 1.55 mg, respectively.The control treatment showed the lowest values, 12.40 mg and 0.76 mg for TFM and BIO, respectively.In relation to ratios, T. asperellum URM 5911 and the control showed lower S/R (2.73 and 2.61, respectively) inferior to the other treatments which value of S/R ranged from 2.85 to 2.95 (Table 1).
In relation to plant height (H) and stem diameter (SD), T. harzianum strain ESALQ 1306 was superior than the other strains (18.65 cm and 15.28 mm), followed by T. asperellum URM 5911 (15.75 cm and 14.38 mm), which was the second best treatment (Table 2).Evaluating head diameter (HD) and number of leaves (NL), T. harzianum strain ESALQ 1306 was superior to the other treatments, providing 13.50 cm and 28.12 leaves.The other strains were superior when compared to the control (10.46 cm and 22.45 leaves), with values for HD ranging from 12.06 to 12.59 cm and NL ranging from 25.31 to 25.75.For fresh and dry mass, T. harzianum ESALQ 1306 was superior to the other treatments, showing TFM of 462.0 g and BIO of 18.92 g, respectively.The control provided the lowest values, 276.98 g and 10.26 g for TFM and BIO, respectively.For ratios, no differences among treatments for SMR, RMR and S/R were no noticed.For productivity, T. harzianum ESALQ 1306 was superior to the other ones (50.20 t ha -1 ), followed by T. asperellum URM 5911, T. harzianum IBLF 006 SC and T. harzianum IBLF 006 WP which provided about 44.23, 42.77 and 41.38 t ha -1 , respectively.All strains were superior compared with the control, which provided 30.18 t ha -1 (Table 2).
Seed germination and seedling vigor are decisive factors for crop production success and presence of hormones, promoters and growth inhibitors are essential for germination physiological process (Ferreira & Borghetti, 2004).
The strains used in this study provided positive and beneficial effects on lettuce seed germination.These results were already expected, according to the information found in literature (Oliveira et al., 2018), since seed germination can be favored by the action of Trichoderma spp.strains.This situation might be related to the fact that lettuce seeds have lower concentrations of reserve substances for germinative process when compared with grainproducing species.Thus, germination process and initial seedling development seem to be stronger influenced by exogenous growth regulators, when associated with microorganisms (Schlindwein et al., 2008).Germination (%), emergence and emergence speed index are promoted when seeds are inoculated with Trichoderma spp.This fact is attributed to growth regulator liberation mechanism and nutrient solubilization (Hajieghrari, 2010;Wesam et al., 2017).
T h o s e s e e d s t r e a t e d w i t h Trichoderma spp.presented length gain.Probably, the most likely mechanism to promote initial growth is via direct, it means, production of hormones or analogues (Wesam et al., 2017).We noted that the seeds showed low or no occurrence of harmful pathogens to germination, refuting indirect initial growth promotion, through harmful pathogens to germination and emergence.
For fresh mass and total biomass accumulations, the strain ESALQ 1306 was also superior to the other treatments.However, the other strains also showed results superior to the control.This can be explained in the study carried out by Chacón et al. (2007), in which these authors state that tomato plants inoculated with T. harzianum showed increased root proliferation and, as a consequence, an increase in sanity and water and nutrient absorption capacity, promoting, as observed by Machado et al. (2012), an increase of fresh mass of several plant parts.
H and SD are productive variables, which allow greater separation capacity among evaluated strains when comparing with HD and NL.According to Trani et al. (2006), NL is a trait directly related to cultivation temperature and photoperiod.Due to this characteristic, Growth promotion and productivity of lettuce using Trichoderma spp.commercial strains the variables HD and NL may be directly related to weather conditions, and the strains may cause only secondary effects on lettuce cultivars.In relation to SD, this characteristic defines seedling's field performance after planting (Souza et al., 2006), due to the ability to form new roots, a fact that is directly influenced by rhizosphere colonized by Trichoderma spp. with consequences on productivity (Carvalho et al., 2011a).
Values of H, SD, HD and NL were similar to the ones obtained in other studies with head lettuce (Table 2).The study carried out by Blind & Silva Filho (2015), for instance, obtained 12.1 to 17.5 cm for HD of head lettuce cv.Balsamo.Santi et al. (2013) obtained SD ranging from 16 to 18 mm and NL ranging from 24.1 to 28.2 in head lettuce fertilized with filter cake.The treatments with Trichoderma spp.resulted, in relation to the control, a gain of 41 to 91% of H, highlighting growthpromoting effect of the used strains.Silva et al. (2015), using different isolates of Trichoderma spp., observed similar effect; the increase in plant height was up to 34% in relation to the control, though.
Although fresh mass of plants is a less frequent component of evaluation in scientific studies on plant growth promotion, the authors highlight the importance to evaluate the strain effect on obtaining root fresh mass since it exploits a greater soil volume and they are also important in plant adaptation in environments with lower quantity of nutrients (Hartwigsen & Evans, 2000).
So, considering that many strains of Trichoderma ssp.can help in nutrient solubilization (Benítez et al., 2004), the authors verified that T. harzianum ESALQ 1306 has potential to act as a nutrient solubilizer.Thus, we verified that the statistical superiority of T. harzianum ESALQ 1306 in laboratory for RFM, SFM, TFM, RDM, SDM and total biomass was also reproduced in field.One explanation for this event lies in the fact that, when one strain of Trichoderma establishes a relationship with rizosphere, plant growth is stimulated.This growth is related to rhizosphere competence, it means, the capacity the strains have to colonize the plant rizosphere and not only to produce growth hormones or analogues (Carvalho et al., 2011a).Carvalho et al. (2015b) stated that, after being applied to a soil in which beans were grown, the strain of T. harzianum ESALQ 1306 was recovered, after the crop cycle, showing populations which ranged from 50 to 100 UFC/g soil.Moreover, not only the ability to colonize roots, but plant growth promotion also depends strongly on the interaction between the isolate and the tested plant species, as well as the conditions of the experiment conduction.
For lettuce productivity, treatment with T. harzianum ESALQ 1306 was considered satisfactory (50 t ha -1 ), since the average of head lettuce productivity using organic fertilizers is higher than 43.1 t ha -1 , according to Sediyama et al. (2016).The other strains had productivity inferior than the productivity of ESALQ 1306, ranging from 41 to 44 t ha -1 .The other strains were superior to the control (30.18 t ha -1 ), though.Similar productivities were found by Yuri et al. (2002), in head lettuce cultivars (Cassino, Legacy, Lucy Brown, Lorca, Lady and Raider) grown in south region of Minas Gerais at 800 m altitude, in typic distrophic Red Latosol, with a commercial production from 28.9 to 42.6 t ha -1 of harvest in March.
Thus, the strains IBLF 006 and URM 5911 could not be ruled out, because not all the efficient bioagents in promoting crop growth have equal efficiency for controlling diseases (Carvalho et al., 2015a,b).
In relation to the ratios in the laboratory experiment, the treatments with T. asperellum and the control were inferior to the other treatments for SMR and S/R; the authors suppose that the reduced biomass allocation to root system in seedlings treated with T. asperellum can be related to water availability to seedlings, since these seedlings were irrigated daily during the test with gerbox and, no water restriction was noticed (Guimarães et al., 2014).Contrary to what was observed in the laboratory, the seedlings did not obtain significant difference in relation to ratios in field experiment.This can be explained by the fact that Trichoderma fungus has differential reaction depending on the environment to which it is submitted (Akrami et al., 2011).
The results of head lettuce growth promotion showed in the treatment with T. harzianum ESALQ 1306 in laboratory were confirmed in the field when productivity and their components were evaluated.The authors recommend the use of strain T. harzianum ESALQ 1306 for growth promotion and head lettuce productivity, since the use of this strain for growing soil treatment provided an increase in productivity.