Soil moisture maintenance methods in cultivation in a greenhouse

Soil water content standardization in agricultural experimentation is essential to mitigate variability within treatments, decreasing experimental errors arising from irrigation management. The aim of this study was to assess soil moisture maintenance methods using sunflower (Helianthus annuus L.) grown under controlled conditions as indicator plant. The experiment was conducted in a greenhouse during the period between August and October 2014, using an oxisol. Treatments consisted of four soil moisture maintenance methods (tensiometer, Irrigas ® , gravimetric method and self-watering system) in eight repetitions, using an entirely randomized design. Variables analyzed were: chlorophyll content, dry matter of the head, leaves, stem, root, and total dry matter, water consumption and water use efficiency for head dry matter yield. Irrigation management based on gravimetric method is sufficient to provide higher biomass accumulation in the crop, consequently increasing plants water consumption.


INTRODUCTION
In tests under controlled conditions, soil water availability is a limiting factor to the growth and development of plants.Thus, the use of soil moisture maintenance methods is necessary to reduce variability within treatments, ensuring statistical tests sensitivity, and consequently, higher reliability to the results (Beltrão et al., 2002).
However, in general, soil moisture maintenance in agricultural experiments, particularly in those conducted in a greenhouse, is done without the adoption of criteria to standardize the water flow restored to experimental units.
Soil moisture variation study requires the use of appropriate methods when monitoring plants water availability (Silva et al., 2012), to prevent excess or drought conditions.
One of the most widely used methods for soil moisture maintenance in scientific experiments is conducted in vases using the gravimetric method.Using vase capacity characterization, also known as pot capacity, soil water volume is kept through the weighing difference of experimental units in a given time interval (Bonfim-Silva et al., 2011).
Taking into consideration the probable soil water *Corresponding author.E-mail: embonfim@hotmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License content variations, other methodologies were proposed for irrigation control in experiments, among which selfwatering system (Silva et al., 2005), Irrigas sensor (Calbo and Silva, 2005;Silva et al., 2014) and tensiometer (Richards, 1942;Libardi, 2005) are highlighted, the last two being identical to the commercially available.
Studies have proven that sunflower is one of the most sensitive crops to soil moisture variation, being used as indicator for plant of water availability level.Nazarli et al. (2010) and Dutra et al. (2012) demonstrated the close relation between soil moisture and crop yield.
Taking into account the different plant experimental treatments conducted in pots, moisture standardization is essential for all experimental plots, to obtain answers between applied treatments and to minimize the coefficient of variation errors during the experimental period.
In this context, it is necessary to establish soil water content maintenance methods that do not affect the yield of plants under experimentation, ensuring higher results reliability.Therefore, the aim of this study was to assess soil moisture maintenance methods using sunflower (Helianthus annuus L.) as indicator plant, under controlled crop conditions.

MATERIALS AND METHODS
The experiment was carried out in a greenhouse at the Agricultural Engineering Graduate Program, Federal University of Mato Grosso, municipality of Rondonópolis, Mato Grosso state.The city of Rondonópolis is located at 16° 27' 52" South latitude and 54° 34' 46" West longitude, at an altitude of 290 m above sea level.
The experimental design was completely randomized, with four soil moisture maintenance methods (gravimetric method, Irrigas sensor, tensiometer, and self-watering system) and eight repetitions, in the period between August and October, 2014.
An oxisol soil collected at 0 to 0.20 m depth in a Cerrado area was used.The soil was passed through a sieve with 4 mm mesh.
Experimental units were represented by plastic pots with 3.18 dm -3 capacity.Base saturation was raised to 60% with dolomitic limestone (PRNT = 80.3%) addition, being kept for 30 days in order to decrease soil acidity.
After limestone incubation, fertilization with phosphorus and potassium was conducted at doses of 150 and 100 mg dm -3 , respectively, using single superphosphate and potassium chloride as sources.
Nitrogen fertilization (200 mg dm -3 ) was split in three applications, at 10, 20, and 40 days after emergence (DAE).Micronutrient fertilization was conducted using a solution containing 1 mg dm -3 of B and Cu, 3 mg dm -3 of Mn and Zn and 0.2 mg dm -3 of Mo, provided by the following sources: boric acid, copper chloride, manganese chloride, zinc sulfate, and sodium molybdate, respectively The cultivar used was Helio 250.Seeds were treated with systemic and contact fungicide.Sowing was conducted by placing ten seeds per pot, and 18 days after emergence (DAE), thinnings were carried out, leaving the two most vigorous plants.
The average daily maximum temperature observed during the experiment was of 36.7°C, and average values of maximum and minimum relative humidities were, respectively, 87.7 and 30.58%.
For irrigation management on soil water maintenance methods, van Genuchten (1980) equation was used according to the determination of the water retention curve in the soil (Dourado et al., 2000), with undisturbed soil samples being taken from experimental units (Equation 1).
Irrigation of all treatments was carried out daily at 7 a.m. and 2 p.m.For soil water content maintenance, an analog scale was used to replace water by the gravimetric method (Figure 1A), and a digital tensiometer was used to measure water tension in the soil (Figure 1B).
The gravimetric method for soil water retention maximum capacity maintenance, or pot capacity (PC), was determined in the laboratory in 3.18 dm 3 pots containing the equivalent of 3.620 kg soil at 3% moisture with mass basis, in ten repetitions.Thus, pots were placed on a support within plastic trays.Water was added up to two-thirds the height of the pots, so that the soil was saturated by capillarity, expelling all the air contained in its pores (Bonfim- Silva et al., 2011).
After soil saturation, pots were removed from the tray and placed on a support to allow free water draining.When pot drainage stopped, deformed soil samples were taken for mass basis moisture determination.
After samples removal, they were immediately weighed to obtain the wet weight.Afterward, they were led to a forced air oven at 105°C for a period of 24 h.After this period, samples were weighed again, and moisture regarding pot capacity was obtained by difference.
With a view to irrigation management during the crop cycle, the volume of water needed to maintain soil moisture at 80% PC was calculated.Thus, water volume and dry soil matter were added to the pot weight, in order to obtain the total weighing value at the time of experimental plots irrigation.
Tensiometry irrigation management was conducted based on soil water tension assessment, using a digital tensiometer (Figure 1B).The water volume to be applied by irrigation was calculated based on the water retention curve in the soil.The ideal water tension in the soil for moisture maintenance was set as 5 kPa.
After tensions had been observed, the corresponding moistures were calculated through van Genuchten (1980) equation.Knowing the current soil moisture and volumetric moisture at 80% PC, water replacement volume was calculated (Equation 2).Water application was conducted manually by measuring the water volume in a semianalytical balance.
× 3180 (2) Where V is the water volume (cm³), the humidity at 80% field capacity, the humidity in each treatment tension (cm³ cm -3 ) and the value 3180 is the soil volume in the pot (cm³).
Irrigation control Irrigas type signaling system was also used to manage soil moisture in the experimental units (Calbo and Silva, 2005).These sensors were manufactured with a wide critical tension range, making the bubbling pressure test necessary, as described in Libardi (2005), in order to standardize porous capsules, selecting those with the same critical tension.In addition, the test was also used to verify if there were bubbling and air A.
B.  Porous capsules were installed in the pot at 15 cm depth, in the wider scope of the root system region.Irrigation time was determined from 20 kPa tension, applying a volume of 250 ml water in each pot, calculated according to Van Genuchten equation.Irrigas system was composed of a sensor (porous capsule) connected through a flexible microtube to a transparent syringe (3 ml), which served as the system signaling device (Figure 2A).
Soil moisture in the self-watering system treatment was maintained with continuous water replacement, in accordance with plant needs.The system consisted of a porous ceramic capsule (filter candle with 0.05 m diameter and 0.07 m height) inserted into the soil through the upper portion of the pot.A flexible microtube (0.005 m inner diameter and 0.001 m wall thickness) connected the ceramic capsule to the constant level reservoir (Mariotte bottle), which was located below the pot (Figure 2B).
Water potential in the soil was established by the water column height between the pot and the reservoir (30 cm), corresponding to a controlled tension of 3 kPa.Thus, continuous crop evapotranspiration ensured water automatic replacement to the soil, making it a self-watering system (Silva et al., 2005).A level scale calibrated in mm and attached to the reservoir enabled water consumption measurement in each experimental unit.
Experiment collection was conducted 69 days after emergence, and chlorophyll content, dry matter of leaves, stem, capitulum, roots, and total dry matter were assessed.In order to obtain the dry matter, samples were packed in paper bags and placed to dry in a forced air circulation oven for 72 h at 65°C, until obtaining a constant weight.
In order to obtain the chlorophyll index, portable chlorophyll index electronic meter (Falker ClorofiLOG® CFL 1030 model) was used.Through chlorophyll meter, reading per leaf was conducted in five middle third leaves located on the main stem of sunflower plants, obtaining a chlorophyll index average per experimental unit.
In addition to these variables, the volume of water consumed during the experimental period and water use efficiency in relation to productivity were also assessed.Results were submitted for analysis of variance when significant to the Tukey test at 5% probability by Sisvar Statistical Program (Ferreira, 2011).

RESULTS AND DISCUSSION
Significant differences were observed between the statistical methods analyzed for chlorophyll index when comparing the different assessment periods.The maximum value of 39.44 was obtained at 22 DAE, decreasing during the other assessments to the minimum value of 28.68 after 69 DAE (Table 1).

Chlorophyll index
Chlorophyll index decrease can be explained by the plant being in the reproductive stage at 34 DAE, period in which photo assimilates were transported to form inflorescences, causing leaf chlorophyll content to decrease.Dordas and Sioulas (2008) found chlorophyll content decrease from the anthesis stage (65.50) to the stage of plant physiological maturity (49.94).The authors attributed this decrease to nitrogen relocation in the plants, with this nutrient being remobilized to form and develop achenes.
Observing the effect of treatments at 69 DAE (harvest), it was found that chlorophyll index in the Irrigas method exceeded by 20.55% the value obtained in the selfwatering system, which provided soil moisture constant maintenance.
Higher chlorophyll indexes observed in the Irrigas system corroborated with Oraki et al. (2012), who reported that, in lower water availability conditions, chlorophyll content increase was observed in all sunflower hybrids analyzed, averaging 49.02 in the treatment maintained at the lowest water content in the soil.
On the other hand, Dutra et al. ( 2012) observed chlorophyll content decrease in sunflower plants of treatments with higher water availability in the soil, resembling the self-watering system.In this case, the higher water content in the soil caused leaf senescence increase by increasing ABA and ethylene concentration, inducing chlorosis increase due to chlorophyll degradation.
In general, assessed productive characteristics highest averages were observed in the gravimetric method, except for the water use efficiency variable, which had higher values in the Irrigas system.

Dry matter
For leaf dry matter, the self-watering system and the gravimetric method promoted the highest averages in relation to tensiometer and Irrigas methods (Table 2).
Sunflower plants showed a positive response to soil moisture maintenance by the self-watering system for this variable.Results may imply that self-watering system constant irrigation provided a condition in which stomata remained open for a longer period of time, establishing higher CO 2 amounts, which is responsible for increasing the dry matter of leaves in plants subjected to this treatment (Nazarli et al., 2010).Canavar et al. (2014) reported the positive influence of soil water maintenance (30 and 60% field capacity) by the gravimetric method in leaf dry matter in an experiment with four sunflower genotypes under controlled conditions at 50 days after sowing.
Sunflower plants showed higher stem dry matter when subjected to the gravimetric method.It was also noted that other methods, such as tensiometer, self-watering system, and Irrigas have not promoted significant differences for this variable (Table 2).Possibly, higher Table 2. Dry matter of leaves -DML, stem -SDM, Capitulum -DMCAP, root -DMR and total dry matter-TDM (g plant -1 ) of sunflowers cultivated with soil moisture maintenance methods at 69 days after emergence (DAE)*.water consumption in the gravimetric method induced sunflower photosynthesis increase, followed by photosynthetic area growth (Hemmati and Soleymani, 2014) and stem biomass production increase.Boareto et al. (2012), while researching evaporation levels variation effect for this crop, reported that lower water content in the soil promoted plant water stress, reducing evapotranspiration due to lower stomatal conductance.This process substantially lowers carbon assimilation and stem biomass production.

Variable
For head dry matter, the lowest average was found in the self-watering system (12.43 g), being 36.35%lower when compared with the value obtained in the gravimetric method (19.53 g).There was no statistical difference between the other methods (Table 2).Soil moisture maintenance by the self-watering system may have influenced in the nitrogen assimilation by plants, influencing biomass accumulation in head formation and development.Zobiole et al. (2010) mentioned that, in head and achene formation, there is photosynthate translocation from accumulated reserves, mainly in sunflower plants leaves and stems, which are dependent on nitrogen availability to the plant.Among soil moisture maintenance methods, it was found that the highest root dry matter yield was observed in the gravimetric method (11.76 g pot -1 ), which was statistically higher than the others (Table 2).
The result can be explained by root distribution in the pots, once plants subjected to the self-watering system focused higher root matter near the porous capsule.Gravimetric method, on the other hand, distributed the water volume evenly in the whole pot, so that the root system exploited higher soil volume, promoting higher root biomass.Paiva et al. (2011) found a significant effect on sunflower plants root system development when they were exposed to water levels in the soil.In addition, the highest root dry matter value was obtained in the treatment equivalent to 75.17% field capacity, corroborating the results of this study, in which the gravimetric method promoted better root development.In sunflower plants total dry matter, the highest yields were observed in the gravimetric method (Table 2).
For Farahvash et al., (2011), the sunflower plants total dry matter decrease is the result of drought during the period of plant growth and development, causing leaf area and photosynthetic process reduction, besides reducing photoassimilates production and leaf, stem, and head development.Nobre et al. (2010) observed 140% shoot dry matter increase when comparing the hypoxic condition with 80% crop water requirement replacement, corroborating the results of this study, in which the highest biomass yields were found in the gravimetric method, where soil moisture was maintained at 80% field capacity.

Water consumption and use efficiency
There was a significant variation in water use by sunflower plants depending on the methodology used to maintain soil moisture.Among methods, the highest water consumption accumulated in the period of 69 days was of 27.39 L in the gravimetric method, which statistically differed from the values verified in the other methods, except for the tensiometer (Figure 3).
It should be noted that, among treatments, only irrigation held in the gravimetric method was not based on water tension in the soil.Instead, it was based on bulk water content, which supported moisture content increase in pot capacity (PC).Souza et al. (2000) attributed soil available water superiority to the gravimetric method based on PC determination, since the soil, after being saturated, only undergoes potential gravitational action.Gravitational potential action power is certainly less expressive than the 5 kPa tension (Porto et al., 2014), present to the other methods based on water tension in the soil, what provided soil water volume increase in PC, and higher water consumption.Flénet and Saraiva (1996), while investigating sunflower crop response to soil water contents, also observed a significant effect of water consumption by plants, reporting higher consumption in the treatment that held the soil under field capacity.
Water use efficiency showed the significant effect to head dry matter when subjected to soil moisture  ) had the highest average, with a 39.76% increase when compared with the self-watering system (0.50 g L -1 ) (Figure 4).This effect may be related to nutrients accumulation by plants subjected to irrigation management with Irrigas sensor, thus influencing head dry matter yield.Thus, higher nutrient concentrations provided further head development.
Results, combined with lower water consumption indicated by Irrigas sensor, caused this method to obtain the best water use efficiency, providing 0.83 g head dry matter production for each 1 L of consumed water.Steduto and Albrizio (2005) have also observed a significant effect of water content variability in the soil on water use efficiency in sunflower plants.They defined water use efficiency as a parameter that relates carbon assimilation by plants to evapotranspiration accumulated at the end of the cycle, constituting a characteristic of great importance in crop biomass gain assessment.
In addition, Naim and Ahmed (2010) reported water use efficiency significant effect for sunflower achenes production in function of different water availabilities in the soil, in which the treatment with the lowest plant water consumption had the highest water use efficiency.

*
Means by the same lowercase letter in the line do not differ by Tukey test at 0.05 probability

Figure 3 .
Figure 3. Water consumption of sunflower cultivated with soil moisture maintenance methods at up to 69 days after emergence.

Figure 4 .
Figure 4. Water use efficiency for head dry matter (WUE) of sunflower cultivated with soil moisture maintenance methods at 69 days after emergence

Table 1 .
Chlorophyll Index of sunflower plants subjected to soil moisture maintenance methods*.Means followed by the same lowercase letter in the column do not differ by Tukey test at 0.05 probability. *