The impact of drought stress on the yields and food value of selected forage grasses

The aim of the study was to compare yields and nutritional value of selected species and cultivars of forage grasses under the optimal moisture conditions and long-term drought stress. The regenerative capacity of plants after dehydration was also assessed. The pot experiment was conducted in years 2009–2010 in IUNGPIB’s greenhouse in Puławy, Poland. Nine cultivars of four species: Dactylis glomerata (‘Amera’, ‘Minora’), Festuca pratensis (‘Skra’, ‘Fantazja’), Festulolium braunii (‘Felopa’, ‘Agula’, ‘Sulino’), and Lolium multiflorum (‘Gisel’, ‘Lotos’) were investigated in well-watered conditions (70% field water capacity – FWC) and under a long-term drought stress (40% FWC). The study showed that stress caused by soil moisture deficiency significantly reduced yields of D. glomerata, F. pratensis, F. braunii, and L. multiflorum. The total yield of dry matter under stress conditions was about 31% lower, compared to the performance achieved on the optimally moisturized treatment. The smallest reduction in dry matter yield under the conditions of water deficit was recorded for D. glomerata, which makes it the most resistant to stress, followed by F. pratensis. The resistance of F. braunii and L. multiflorum to stress was similar and significantly lower. There was a various response of different grasses to the water stress. On the basis of the value of the DSI (drought susceptibility index), the tested cultivars were ranked depending on the sensitivity to drought, starting with the most resistant cultivar: ‘Minora’, ‘Skra’, ‘Fantazja’, ‘Amera’, ‘Sulino’, ‘Agula’, ‘Gisel’, ‘Lotos’, and ‘Felopa’. The digestibility of dry matter and nutrient value of the grasses depended on both the level of soil moisture and grass species. Under the water stress, the digestibility and protein value increased compared to the control objects. Lolium multiflorum and F. braunii had the best nutritional value, while D. glomerata – the weakest.


Introduction
In Poland there are more and more frequent periodic water shortages on large areas of country, which cause large losses in agricultural production [1][2][3][4].In the last decade, severe droughts occurred in the years of : 2005, 2006, 2008, 2010, 2011, 2013, and 2015 [5].Droughts can occur at different times of the year, with varying intensity, duration and scope, but most commonly begins in the spring and summer (65% of cases) [5].
In the system of agricultural drought monitoring, meteorological conditions causing drought are determined by climatic water balance (CWB).According to Doroszewski et al. [6], after 2000 in Poland, in spring and early summer, CWB values have been getting lower and lower, which means that droughts are becoming more and more severe.This coincides with a period when plants have the highest demand for water, which leads to a number of negative environmental and economic consequences.
Forage grasses have a high demand for water.Their daily need is from 0.5 to 3.0 liters per m 2 , and the amount of water transpired per year from 1 m 2 of turf grass may reach 1000 L [7].According to Łabędzki [8], summer drought accompanied by high temperature, can cause a decrease in grass yield by about 30%.The reduction of the yield caused by a given stress factor is a good indicator of plant resistance to this stress [9].Regrowth after dehydration is another important property of grasses.The plants switch from the state of dormancy caused by drought to the state of growth after rehydration of their habitat.Regrowth after a drought often reveals differences among different species and cultivars in terms of the resistance to stress [7,10].Stress affects also the chemical composition and nutritional value of forage grasses.According to some authors [11][12][13], it increases the total protein content and lowers the content of crude fiber, while according to others [14], total protein concentration decreases under the conditions of lower soil moisture.The contents of protein and fiber are the basic elements of the chemical composition of grasses, affecting the protein value and digestibility of feed.
The aim of the study was to compare yields and nutritional value of selected species and cultivars of forage grasses under the optimal moisture conditions, as well as under the conditions of long-term drought stress, and to assess the regenerative capacity of plants after dehydration.

Material and methods
Two-factor pot experiment was performed in years 2009-2010, in a greenhouse of the Institute of Soil Science and Plant Cultivation -State Research Institute in Puławy, Poland (Lubelskie Voivodeship).The completely randomized block design method, with four replication was performed.Nine cultivars of grasses, belong to four species, were tested: Dactylis glomerata L. ' Amera' and 'Minora' , Festuca pratensis L. 'Skra' and 'Fantazja' , Festulolium braunii (K.Richt.) A. Camus 'Felopa' , ' Agula' and 'Sulino' , Lolium multiflorum Lam.'Gisel' and 'Lotos' .Grasses were evaluated at two levels of soil moisture: 70% field water capacity (FWC; optimum soil moisture) and 40% FWC (drought stress) [15].In order to maintain the appropriate soil moisture, water losses were made up on a daily basis, to achieve a specified weight of the pot with soil.Soil moisture was differentiated in the spring, 8 weeks after sowing and lasted the whole vegetation season (long-term stress).In the second year of growing, the observations were continued only in the first, spring regrowth.Regenerative abilities of grasses were evaluated after a 10-day drying period, i.e., a complete suspension of all watering until the drying of approximately 50% of shoots of the most sensitive cultivar.
The seeds were sown in Mitcherlich pots in 7 of April 2009, on a lessive soil from arable layer (0-30 cm).Three seeds of each grass were sown at 30 points per pot.After the emergence of plants, only 15 of them were left.The collection of plants was carried out three times during the growing season.The contents of available nutrients (mg per 100 g soil) were: phosphorus 32.0, potassium 12.3, and magnesium 5.1.Soil pH KCl was 6.2.The pots were fertilized at doses (g pot −1 ): 3.6 N in three rates, 1.0 P, 1.5 K, 0.5 Mg in the form of solutions: NH 4 NO 3 , KH 2 PO 4 , K 2 SO 4 , and MgSO 4 × 7 H 2 O.The yield of plants and the drought susceptibility index (DSI) were determined, which allowed for dividing cultivars into groups with different tolerance to water shortages in the soil.The value of DSI index was estimated based on the formula [16]: ] − 1, where: D n -dry matter yield of the plants subjected to stress; K n -dry matter yield of the plants grown under optimal moisture; D x -the mean dry matter yield of all treatments under drought conditions; K x -the mean dry matter yield of all optimally moisturized treatments.A low value of DSI index indicates a relative resistance to stress conditions, while a high -a low resistance to stress.
Chemical analyses of plant material were based on mean treatment samples of grass dry matter from the first regrowth in the first and second year, due to its highest quantitative and qualitative changes and the importance in the annual yield, at the Main Laboratory of Chemical Analysis of IUNG -PIB in Puławy.The contents of dry matter (by weighing method at 105°C) and of total N (by the flow spectrophotometric method) were determined.They were used as a basis for calculating the contents of total protein (BO = N × 6.25), crude fiber (by conventional method), and the digestibility of dry matter (by enzymatic method).Protein and energy values of the feed obtained at the optimal moisture conditions and drought conditions were calculated according to the French system INRA [17], using WINWAR 1.3 program cooperating with the INRA program.The results were statistically analyzed with the use of the analysis of variance using Statistica v. 10.0 program.Tukey's multiple comparison test was used to compare differences between the means for main effects (factors), while confidence intervals for the means of Fisher's least significant difference (LSD) test (α = 0.05) were used to compare the means from the subclasses (interactions).

Results
Soil moisture was an important factor affecting the dry matter yield of the species and cultivars of forage grasses.Under optimum soil moisture, the best yields were obtained for L. multiflorum 'Gisel' and 'Lotos' (Fig. 1).A significantly lower total yield of dry matter was obtained for hybrid F. braunii, but a greater efficiency was found for 'Felopa' and ' Agula' cultivars as compared to cultivar 'Sulino' .Festuca pratensis 'Fantazja' and 'Skra' yielded at a similar, average level.Dactilis glomerata 'Minor' was found to have the lowest efficiency.Grass cultivars reacted with a significant yield decrease to the limited soil moisture (average by 31.1%).The smallest decrease of the total yield was recorded for D. glomerata 'Minor' , while the biggestfor F. braunii 'Felopa' and ' Agula' (accordingly by 37.8 and 34.5%) and for L. multiflorum 'Lotos' and 'Gisel' (accordingly by 35.0 and 34.4%).Analyzing individual regrowths, it was found that the stress caused by the shortage of water in the soil limited the yield of grasses in the first regrowth the least (average by 22.7%), while significantly more in the second and third regrowth (accordingly by 35.3 and 34.4%) compared to the optimally moisturized treatments (Tab.1).
The reaction of grasses to the stress caused by water deficits in the soil was dependent on the species and cultivar.The group resistant to drought (DSI < 1) included D. glomerata and F. pratensis, while the sensitive group Tab. 1 Relative dry matter yield of grass species and cultivars in relations to control object in the first year (%).-L.multiflorum and F. braunii (Fig. 2).Among the tested D. glomerata cultivars, 'Minora' was more stress-resistant than ' Amera' .Among the F. pratensis cultivars, 'Skra' was more resistant than 'Fantazja' .Festulolium braunii hybrid showed a diverse sensitivity to drought.The cultivar 'Sulino' was the least sensitive to stress, while 'Felopa' -the most.Lolium multiflorum cultivars had a similar resistance to drought.Taking into account the values of the DSI index, the tested cultivars can be ranked in accordance with their sensitivity to drought in the first year of utilization, starting from the most resistant cultivar: 'Minora' , 'Skra' , 'Fantazja' , ' Amera' , 'Sulino' , ' Agula' , 'Gisel' , 'Lotos' , and 'Felopa' .

Regrowth
Regenerative capacities of the tested species and cultivars of grasses were investigated in the second year of growing in the first regrowth after a 10-day period of drying.Dry matter yields were calculated in absolute values (Fig. 3) and in relative values, as a percentage of the dry matter yield of the tested plants as compared to the control (Tab.2).The yielding ability of grasses after a period of complete abandonment of watering was the highest in the case of F. pratensis 'Skra' , followed by D. glomerata 'Minora' , while in the case of the other grasses, it was significantly lower.Hybrid F. braunii 'Felopa' showed the lowest regeneration capabilities.The highest values of the coefficient of variation were recorded for L. multiflorum cultivars 'Gisel' and 'Lotos' , while the lowest for F. braunii ' Agula' and 'Sulino' .None of the tested cultivars was able to recreate the conditions to the level of the plants not subjected to stress.
Nutrients content in dry matter yield of first regrowth was dependent on the soil moisture and the species of the grass (Tab.Tab. 2 Yielding capacity of grasses species and cultivars after drying-up period.

Coefficient of variation (%)
Dactylis growing, as compared to the control.A large variation in the content of this component was recorded also between species of the grass.Under the optimal soil moisture and under stress, the largest amount of crude fiber was found for D. glomerata, and the lowest for L. multiflorum and F. braunii hybrid, which were similar in this aspect.In the second year of growing, more crude fiber was recorded for D. glomerata, followed by F. pratensis and L. multiflorum, while the least for F. braunii.The level of soil moisture affected also the digestibility of grass dry matter.Under drought stress, the value of this indicator was significantly higher, in the first year on average by 3.9%, while in the second on average by 5.0% compared to the optimally moisturized objects.Moreover, this feature was also more closely related to species than to cultivars.Regardless of the level of soil moisture, in the first year of growing, the highest, similar digestibility was recorded for L. multiflorum and F. braunii, while significantly lower -for D. glomerata.Festuca pratensis had a medium value of this ratio.In the second year of growing, a significantly higher dry matter digestibility was found for L. multiflorum, F. braunii, and F. pratensis than for D. glomerata.
The nutritional value of roughage in the diet of ruminants is determined by, besides digestibility, its protein and energy value (Tab.4, Tab.5).The protein value expressed in PDIN, PDIE, and PDIF was higher under reduced soil moisture, whereas significant differences occurred mainly in the first year of growing.In contrast, the energy value of the studied grasses expressed in nutrient units of milk (UFL) and livestock production (UFV) had higher values under stress conditions, but statistically significant differences were not proven.The nutritional value of biomass was also more dependent on a species than on a cultivar.In the first year of growing, the highest protein value was recorded for F. pratensis and F. braunii, and energy value for F. braunii and L. multiflorum, while in the second year, F. pratensis, L. multiflorum, and F. braunii had a similar PDIN, PDIE, and PDIF values, while UFL and UFV values were the highest with F. braunii.The lowest nutritional value in both years of vegetation was recorded for D. glomerata.

Discussion
Fodder grasses reacted to stress conditions caused by a moisture deficiency in the soil with the reduction of their yields, due to inhibition of the processes of plant development and growth.The results of the research have confirmed that D. glomerata, F. pratensis, F. braunii, and L. multiflorum had significantly lower dry matter yields (average by 31%) in condition of water stress.The smallest reduction in dry matter yield under stress was recorded for D. glomerata and F. pratensis, while significantly higher for F. braunii and L. multiflorum.Using the yield reduction as the criterion for measuring the level of stress resistance, it was assessed that D. glomerata was the most resistant to long-term drought, followed by F. pratensis.The resistance of F. braunii and L. multiflorum was much smaller and similar.It showed that, in terms of sensitivity to water deficits in the soil, the hybrid F. braunii was more similar to L. multiflorum than F. pratensis.Olszewska et al. [18] showed that a long-term water stress (35% FWC) caused a significant reduction in the yield of the three species of grass.The smallest reduction of dry matter yield was recorded for Lolium perenne (46%), higher for D. glomerata (57%), and the highest for F. pratensis (68%).Madziar and Latanowicz [11] found, however, that regardless of soil moisture, D. glomerata yielded the best, Phleum pratense worse, while F. pratensis the least.In field conditions, irrigation of forage grasses significantly increases yields in dry years.According to Rumasz-Rudnicka [19], irrigation of Lolium westerwoldicum increased its green matter yield by 17%.According to Norris and Thomas [20], withholding the irrigation of Lolium sp. for 10 days before mowing, reduced its yield by 20%, while the abandonment of irrigation for 6 weeks -by 45%.The current results have conformed previous observations of low drought resistance of F. braunii hybrid [21].It was observed that limited rainfall in May resulted in the reduction of annual yields of F. braunii 'Felopa' by 15% in relation to the achievement in the optimal previous year.The sensitivity of this hybrid to the lack of moisture in the soil has also been reported by Wilman [22], Łyszczarz et al. [23], Borowiecki [24], as well as Gutmane and Adamovich [25].However, there was Tab. 4 Dry matter digestibility and energy and protein value of grass cultivars depending on soil moisture in the first cut in the first year of vegetation.Explanations: PDIN -protein digested in the small intestine supplied by rumen-undegraded dietary protein plus protein digested in the small intestine supplied by microbial protein from rumen-degraded protein; PDIE -protein digested in the small intestine supplied by rumen-undegraded dietary protein plus protein digested in the small intestine supplied by microbial protein from rumen-fermented organic matter; PDIF -protein digested in the small intestine; UFL -feed unit for lactation; UFV -feed unit for maintenance and meat production.

Species
Tab. 5 Dry matter digestibility and energy and protein value of grass cultivars depending on soil moisture in the first cut in the second year of vegetation.no confirmation for the research results of Thomas and Humphreys [26], who claimed that this hybrid was as draught resistant as F. pratensis or Domański and Jokś [27] who showed that under soil moisture deficits, F. braunii hybrid yielded at a similar level to Festuca arundinacea.Growth inhibition under drought is a typical defense reaction among plants.Water deficit in the soil leads primarily to the reduction of water potential of plant shoots and to the stimulation of the growth of the root system.A direct reaction of plants is to change the method of distribution of assimilates, which in turn lowers the yields of biomass [28,29].

Species
The selection of proper cultivars is, besides habitat and agrotechnical conditions, one of the yield determining factors [30].The obtained results showed that the tested cultivars responded differently to the shortage of water in the soil.Dactylis glomerata 'Minora' was more resistant to stress than ' Amera' , F. pratensis 'Skra' -than 'Fantazja' , and F. braunii 'Sulino' -than 'Felopa' and ' Agula' .The reaction of L. multiflorum 'Gisel' and 'Lotos' was similar.The studies conducted by Szoszkiewicz et al. [31] also showed a different reaction of grass cultivars to water stress.Under optimal soil moisture, the highest yields were obtained from late cultivars of D. glomerata 'Satra' and F. pratensis 'Westa' , while under water stress (40% FWC) -from semi-early cultivar of D. glomerata ' Amera' and early cultivar of F. pratensis.According to Olszewska [32], among the tested F. pratensis cultivars, 'Skra' had a better efficiency both under optimal soil moisture and water scarcity, compared to 'Skawa' , but significant differences under stress conditions have not been proven.In other studies, the same author recorded a higher yield of D. glomerata 'Dala' compared to ' Areda' , regardless of the level of soil moisture [33].According to Kochanowska-Bukowska [34], among the cultivars of D. glomerata, ' Astera' yielded the highest, ' Amera' and 'Bepro' significantly lower, while 'Potomac' the lowest.Our results have confirmed prior literature reports concerning different reactions of individual cultivars to water stress.Dziadczyk [9] suggested that this difference results from genetic conditioning of the cultivars of a given species to a particular stress.
The cease of drought, usually after soil hydration, leads to the start of plant regeneration process.According to Kemp and Culvenor [35], it is more important to plants than maintaining growth during drought.In this study, after a 10-day period of a complete abandonment of watering, there was no complete regeneration of grasses, i.e., restoration of their prior-drought state.Festuca pratensis 'Skra' and D. glomerata 'Minora' regenerated the best (fielding abilities after stress -accordingly 91 and 89%), while F. braunii 'Felopa' -the worst (54%).According to Żurek [36], L. perenne is a species capable of relatively high recovery after the most stressful conditions.After a complete dryout, the cultivars of this species regenerated achieving approximately 59% of the total health of plants grown under control conditions.Withering of leaves during the prolonged lack of water is very important for plants, as it allows for the transfer of proteins, fats and other macromolecules to other organs, such as the buds of young leaves, flowers or seeds, from which the plant will be able to renew itself after the cease of drought [37,38].According to Volaire and Leliévre [39], grass regeneration process depends largely on the density and the degree of the regrowth of the shoots subjected to long-term water shortages and on the growth of new shoots.Leaf buds are the key organs which determine the survival of grasses of water shortage periods.They can tolerate much lower values of osmotic potential than fully developed leaf blade, and they initiate subsequent regrowth of plants.In fact, the hydration of young tissues and elongating leaf blades and sheaths occurs the fastest, while in the case of mature leaves, it is much slower [37].According to Carrow [40,41], withering of grass leaves, which is a symptom of the gradual degradation of chlorophyll, is considered a good indicator plant resistance to drought conditions.A diversified drying out and regeneration are also crucial for the evaluation of breeding lines because of the great diversity of genotypes.
The sensitivity of plants to environmental stress is determined by the value of drought sensitivity index (DSI).Determination of DSI index allows for the comparison of sensitivity to water stress of different grass genotypes for the selection of the most valuable ones, e.g., for further breeding works and introduction of new, improved cultivars into practice.This method of estimating the quantitative indicator of drought tolerance is simplified, because it does not take into account the interaction of the drought conditions with the development stages or important physiological aspects of plants, especially transpiration.It provides, however, the general information on the basis of which it is possible to rank the cultivars in terms of their yields under limited water supply.Dactylis glomerata 'Minora' was the most resistant to drought, while F. braunii 'Felopa' -the least.The evaluation of drought resistance of spring wheat cultivars has been performed by, among others, Grudkowska et al. [42] and also Bahar and Yildirim [43].
The factors included in our studies differentiated organic nutrients content and nutritional value of grass.Soil moisture had a greater impact on crude protein and crude fiber than the grass cultivar.Under drought stress, all the species had more total protein, but less crude fiber in comparison to the optimally moisturized treatments.These results are consistent with the results of other researchers who found an increase in protein content and a reduction in crude fiber content of D. glomerata, F. pratensis, and P. pratense under water deficit in the soil [11,31,32].Also in the study of Trzaskoś et al. [44], there was an increase in total protein content in dry matter of grass sward under dry conditions.According to Zimont and Pawlak [45], water stress contributed to the reduction of dry matter yield and the increase of total protein content of L. multiflorum, while it did not initially affect the protein yield.Yet, after further drying of the soil, protein yields rapidly decreased, which indicated the deterioration of animal feed under drought.

1 )Fig. 1
Fig. 1 Decrease of dry matter yield (sum of three cuts) of selected forage grasses under conditions of drought stress in the first year.Different letters on bars represent significant differences among treatments (LSD test, p < 0.05).

Fig. 2 Fig. 3
Fig. 2 Drought susceptibility index (DSI) for grass cultivars under conditions of water deficit.
Content of total protein, crude fiber and digestibility of dry matter of grass cultivars depending on the soil moisture in the first cut.