Influence of Fertilizers on the Biodiversity of Semi-natural Grassland in the Eastern Carpathians

This investigation examines the influence of fertilization with organic and mineral fertilizers on the biodiversity of grasslands containing Festuca rubra, Agrostis capillaris and Nardus stricta. Permanent meadows were studied in terms of production of food, and of biodiversity. The current strategy of using organic fertilizers has raised concerns about resource conservation and environmental protection. The increase in the number of species is due to the fertilizers that have been applied leading to changes in the soil fertility status. This change in soil fertility has allowed other mesotrophic and eutrophic species to become established in fertilized meadows. In Romania, meadows belonging to this category occupy an area of approximately 1,600,000 hectares and have relatively low production rates. The experiment was located at Pojorata, Suceava County (Romania), in two different natural grasslands that had different floristic compositions. Manure improved the growth of a number of species, especially in the “plants from other botanical families” category, because of the pool of seeds that it contains. Using a management system based on fertilization with small amounts of organic and mineral fertilizers can help preserve the biodiversity of these meadows. The results of this study, in an area considered representative for large parts of the mountainous areas of Romania, indicated that fertilization treatments were able to maintain a high species diversity.


Introduction
Semi-natural grasslands, which are traditionally used as forage for ruminants, are an important type of land use in Europe, covering more than a third of the European agricultural area (Pacurar et al., 2012).Fertilization with manure are considered an appropriate management technique to conserve biodiversity value (Assaf et al., 2011;Chapin et al., 2000;Kesting et al., 2009;Louault et al., 2005;Peeters et al., 2004).Several management factors may affect the biodiversity of these grasslands, including fertilization, overseeding, grazing and cutting management (Duru et al., 2010;Lehman et al., 2000;Mauz and Rémy 2004;Paine et al., 1999;Pasho et al., 2011;Samuil et al., 2012a;Sirbu et al., 2012).Grasslands are an important forage resource in Romania, but poor management during the last few years have led to their present state of degradation.
The greatest reduction in permanent grassland productivity has been caused by unfavorable weather conditions and poor management (Samuil et al., 2012b).An increase in the productive potential of these meadows can be achieved by fertilization with different rates and types of organic fertilizers (Hopkins et al., 1990).Research conducted to date has demonstrated the positive effects of organic fertilizers on lawns (Hacala and Pflimlin, 1994;Ziegler, 1997).If regularly and sensibly applied, organic fertilizers can fully substitute for chemical fertilizers ( Jeangros et al., 1994( Jeangros et al., , 2002)).
Comparative studies that investigated the effects of different management practices on grasslands have demonstrated shifts in species diversity and in the composition of plant functional groups (Baudry, 2004;Dale et al., 2012;Hopkins et al., 1999).Each permanent grassland sward is a unique mixture of species and growing stages and this complexity makes it difficult to characterize and understand their feed value (Duru et al., 1998).Floristic composition influences the nutritive value of permanent grasslands because of the differences in the chemical composition and digestibility of individual species and also because of variations in the growth rate of different species (Dale et al., 2012;Duru et al., 1998).
In this study, an experimental approach was used to evaluate the effects of management treatments on the biodiversity for two semi-natural grasslands in the Oriental Carpathian Mountains.The two following key questions were addressed: (i) are there any changes in plant species composition and in the functional groups of plants under the different fertilization treatments and (ii) what are the temporal trends over a period of six consecutive years?

Study area
This paper presents the results of two experiments located at Pojorata, Suceava County (Romania), on two different natural grasslands with different floristic compositions.The first experiment took place on a meadow com-

Experimental treatments
The experiments were subdivided into plots and were replicated four times.Each plot was 4 m × 5 m in size.They were subjected to the fertilizer treatments shown in Tab. 1.
The chemical composition of 1000 kg of manure was 5.19 kg N, 2.83 kg P and 6.72 kg K.The manure was applied during the autumn season, but the mineral nitrogen was applied in spring before vegetative growth began.
The Used Apparent Coefficient (UAC) was calculated so that the effects of the different types and combinations of organic and mineral nitrogen fertilizers could be compared.The UAC gives an indication of the effective use of organic nitrogen on a short-term basis.The UAC coefficient varies depending on the time of application and the type of vegetation cover and ranges in value from 0 to 70%.The UAC of the nitrogen equivalent for mineral fertilizer is the amount of nitrogen used by the plant.The results obtained in this experiment were based on a UAC value of 0.4 for manure applied annually, 0.45 for manure applied every 2 years and 0.55 for manure applied every 3 years (Bodet et al., 2001).

Analyses and statistical interpretation
The mean indicator values for light (L), temperature (T), soil moisture (W), soil reaction (R) and soil trophicity (Tr) were calculated based on the number of species from a given category.Species indicator values followed the scales of Ellenberg et al. (1992).In order to calculate the biodiversity indicators, surveys were conducted at posed of Agrostis capillaris and Festuca rubra (experiment 1), and was located at an altitude of 707 meters on land with a slope of 20%.The second experiment took place on a meadow containing Nardus stricta (experiment 2), located at an altitude of 727 meters on land with a slope of 28%.
Climatic conditions in the area are characterized by an average temperature of 6.3°C and 708.2 mm total annual precipitation.Between April and September, the average temperature is 12.8°C and rainfall amounts to 513.5 mm.In terms of climate, the areas are located on the northeast extremity of the European Central Province, with a temperate-moderate-continental climate that is also influenced by the eastern continental climate and the northern boreal climate.The species of plants found in the two experimental areas were sub-heliophile, micromezotermophile, mezophile, low acid-neutrophile and oligo-mezotrophile plants.
Permanent grassland occupies 156 000 hectares in the area where this research was conducted, with different plant distributions depending on altitude, slope, temperature and precipitation (Fig. 1).
The soil type was Typic Dystrudepts, according to the American System or Dystric Cambisol according World Reference Base for Soil Resources (WRB-SR).The soil had a pH (measured in H 2 O) of 4.7 and the average nutrient contents were: total N content, 1.3 g kg -1 ; P content, 0.33 g kg -1 and K content, 14.6 g kg -1 .The values for the available P and K in the soil are considered low in phosphorus, but adequate for potassium.five points for each experimental parameter.Presence/absence data and plant cover were recorded.The cover of all vascular plant species was visually estimated in each plot based on the Braun-Blanquet methodology (Cristea et al., 2004).In mid-June 2011, in order to eliminate edge effects, relevés were taken in the center of each 4 m ×5 m plot which covered an area of 2 m ×3 m.
The results obtained by the Braun-Blanquet methodology were transformed in cantitative value.The mean cumulative coverage of the plants was calculated from the abundance dominance values of speciesafter they had been converted to percentages (median percent values of the corresponding Braun-Blanquet classes).These results were used to calculate the Shannon Weaver index, the Simpson index and the Eveness index.Species richness was a count of the number of plant species present in a sample plot (Cristea et al., 2004) and when used in combination with diversity indices, revealed further trends in plant diversity that the more complex indices could not reveal.
The statistical data was analyzed by SPSS using ANO-VA and the Lowest Significant Difference test (LSD).

Results and discussion
Botanical composition was very weak and was represented by species with low forage values (Tab.2, Fig. 2).A total of 35 species were identified at the beginning of the experiments, of which 12 belonged to the Poaceae family, seven belonged to the Fabaceae family and 16 plant species belonged to other families.For each of these species, the ecological indicators were specified.
In the first experiment, 65.8% of the species had a high percentage tolerance to temperature and 63.2% had a high tolerance to soil acidity.In the second experiment, 61.9% of the species had a high percentage tolerance to temperature and 57.1% to soil acidity.
Constancy is an expression of a species relative to the area taken up by each species depending on the experimental variants assessed and provides information on the degree of fidelity.It can be seen that over 60% of the species had a high and a very high constancy, (classes IV and V; Fig. 3).
The application of mineral and organic fertilizers to the Agrostis capillaris L. with Festuca rubra L. grassland and the Nardus stricta L. grassland produced significant changes within the sward, depending on the applied treatments.However, the floristic composition in both experiments was quite similar.Analysis of the biodiversity parameters highlighted the fact that the number of species increased across all the variants compared to the control when fertilizers were applied (Tab.3).
In experiment 1, the number of species increased from 15, in the control plots, to 17-26 in the fertilized plots.The Shannon index increased from 2.504 in the A 0 b 0 fertilized plots (control) to 2.611-2.855 in the fertilized plots.Shannon evenness values were 0884 in the A 0 b 0 plots and 0.875-0.932 in the fertilized plots.The Simpson index was A-manure application rate: A 0 -0 t ha -1 , A 1 -10 t ha -1 , A 2 -20 t ha -1 , A 3 -30 t ha -1 ; B -length of time the manure was applied: b 0 -no manure, b 1 -annually, b 2 -every 2 years, b 3 -every 3 years; C -mineral nitrogen application rate each year: c 1 -30 kg ha -1 , c 2 -50 kg ha -1 ; UAC-apparent coefficient of use (0•4 for manure applied annually; 0•45 for the manure applied every 2 years and 0•55 for the manure applied every 3 years) Numerous studies have revealed positive relationships between biodiversity and ecosystem function (Chytrý et al., 2009;Ganatsas et al., 2012, Reiss et al., 2011).Grassland species richness was linked to the type of fertilizers 0.100 in the A 0 b 0 plots and 0.075-0.098 in the fertilized plots.
In experiment 2, the number of species increased from 14 in the control plots to between 15 and 19 in the fertilized plots.The Shannon index was 2.534 in theA 0 b 0 plots and between 1.936 and 2.355 in the fertilized plots.The Shannon Evenness values were 0.914 in the A 0 b 0 plots and Tab. 2. Plant composition and ecological characteristics of the identified species (after Ellenberg et al., 1992;Kovács, 1979

Conclusions
The results showed that there were changes in the number of plant species and in the functional groups of plants under the different fertilization treatments.The increase in the number of species could have been caused by improved soil nutrient content after fertilizer application and/or by the species brought in with the applied manure.Using a fertilization management program based on small amounts of organic and mineral fertilizers could contribute to the conservation of biodiversity in these grasslands.The results of this study, in an area considered to be regionally representative of large parts of the mountain areas of Romania, indicated that fertilization treatments could maintain high levels of species diversity.applied and to the intensity that animals grazed the grassland resource on a year-round basis (Cao et al., 2011;Hejcman et al., 2010).
The increase in the number of species is due to the application of fertilizers, which changed the soil fertility status.This change in soil fertility allowed other mesotrophic and/or eutrophic species to establish themselves in the fertilized plots.Manure fertilizer caused the greatest increase in the number of species, especially in the "plants from the "other botanical families" group because of the pool of seeds that it contains.This study demonstrated that low doses of manure, applied at different intervals, together with low doses of chemical fertilizers, significantly contributed to the increase in the number of species present in the vegetation cover.

Fig. 1
Fig. 1 Natural conditions of the permanent grassland research areas used in this study

Fig. 3 .
Fig. 3. J-shaped curve for Raunkiaer interfered from the constant value classes of the species found in the second natural grassland (I-V represent the classes of the constancy: I for the plant with low presence <20%; V for the plant present with high presence >80%)

Fig. 4 .
Fig. 4. Relationships between treatments and some diversity indexes (species richness, Shannon diversity index, Shannon evenness and Simpson index) Tab. 1 Fertilization methods applied and the quantities of nutrients supplied to the treatments Tab. 3. Diversity parameters measured in 2010 and 2011 ns -non significant, *p<0.05,**p<0.01