Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY
MEDICINE OF BANAT TIMISOARA
FACULTY OF AGRICULTURE
Biologist BOSTAN CRISTIAN
ALLELOPATHIC ASPECTS IN THE
PERENNIAL GRAMINAES
Scientific:
PROF. DR. DR.H.C. ING. ALEXANDRU MOISUC
TIMIŞOARA
2009
I
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
ABSTRACT
The PhD thesis entitled "Allelopathic aspects in the perennial graminaes" elaborated
under the guidance of Prof. Dr. ALEXANDRU MOISUC is composed of two parts bibliographic study and personal research - structured in 8 chapters, to which discussions and
general conclusions were added. PhD thesis contains 63 tables, 53 graphics and figures (images).
PART I. BIBLIOGRAPHIC SINTHES
The bibliographic study contains 3 chapters and is included in 61 page.
CHAPTER 1. General aspects concerning the allelopathic phenomenon refers to data about
the history of allelopathy, the role of allelopathy in a phytocoenosis , methods of diffusion of the
allelopathic substances, general considerations on allelopathic compounds, allelopathy caused by
plant residuals, the allelopathy at the invasive plants, the impact of allelopathy in agriculture and
the allelopathy role in sustainable agriculture.
The observations on the allelopathic interaction between plant species have a history of
centuries, but the transition from observation to scientific certainty has been achieved relatively
recently.
The structure and the way of action of allelochemical compounds are diverse. It is
important to identify at first the classes of allelopathic compounds and also the concentrations
respectively the mechanisms by which they reach the environment, so that we can form a general
view of their function as precise as possible.
The allelopathic phenomenon is a cause-effect relationship between substances with
allelopathic potential and the physiological response of the target plant. The allelopathic
mechanism can be characterized as a concatenation of biochemical, and physical processes, the
allelopathic messenger and acceptor specie.
II
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
The huge interest that is manifested by the international community on allelopathic
phenomenon is largely due to the fact that the allelopathy strongly plead for the idea of
sustainability in agriculture and thereby for the increase of food product quality, for reducing the
environmental pollution and the risk of disease due to the use of various synthetic substances
(herbicides, pesticides, chemical fertilizers, etc.), for the reduction of erosion and also for finding
alternative solutions to these problems, solutions that nature gives us generously.
CHAPTER 2. The description of the studied perennial graminaes includes data about the
importance of perennial graminaes as well as biological and morphological characteristics of
Dactylis glomerata, Festuca rubra, Lolium perenne and Poa pratensis.
CHAPTER 3. Experimental conditions
deal with the pedological and agrochemical
characterization of the used soil, the main features of the regional climate and the microclimatic
conditions in the studied period 2006-2008.
CHAPTER 4. The objective of the research. Establishing objectives and choosing methods
Researches aimed at the study of the allelopathy’s way of action by laboratory direct
methods and indirect methods (observations "in vivo").
Objectives were:
• the study of the behavior of some perennial graminaes (Lolium perenne, Dactylis
glomerata, Festuca rubra and Poa pratensis) in mixtures at different distances between the rows.
• the determination of the effect given by standardized extracts obtained from the studied
species on each of the studied perennial graminaes .
Materials and methods
The experiments were performed both in the green house of U.S.A.M.V.B. Timisoara
and in laboratory. For all the experiments the used material was represented by four species of
perennial graminaes Lolium perenne, Dactylis glomerata, Festuca rubra and Poa pratensis.
The methods were:
III
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
• Obtaining and analysis of vegetal extracts. The extraction of phenolic
compounds [Djurdjević et al., 2005, Provan et al., 1994, Lowry et al., 1993];
• The testing method of seeds germination;
• The determination of chemical composition at the studied species;
• The analysis of total phenolic compounds by using the Folin Ciocâlteu method;
• The qualitative and quantitative analysis of phenolic acids by using the HPLC
technique (high performance liquid chromatography);
• The determination of ergovaline in the vegetal material. [Morrie et al., 1994,
Davies et al., 1993; Rottinghaus et al., 1991];
• Chromatographic analysis of ergovaline purified extracts;
• Methods of calculating and interpreting the results.
PART II – A. PERSONAL RESEARCH
The personal research includes 5 chapters, extended on 123 pages, which include 61
tables and 42 figures and original images.
CHAPTER 5. Seed germination and perennial graminae plant growth under the influence
of extracts.
Chapter V attempts to highlight the possible effect on seed germination, given by the
lolinic alkaloids found in low alcoholic extracts (20% alcohol), obtained from the four species of
perennial graminaes Lolium perenne, Dactylis glomerata, Poa pratensis and Festuca rubra.
For this purpose, the seed germination percentage of perennial graminaes was tested in
order to determine to what extent the germination capacity is inhibited by lolinic alkaloids from
the obtained extracts.
The obtained extracts were applied in three different concentrations; from high to low
concentration in order to observe to what extent the increase of the allelochemicals level inhibits
seed germination.
IV
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
The lolinic alkaloids content in extracts was as follows:
In the extract of Lolium perenne: Variant 1 - Concentrate = 0.668% alkaloids; Variant 2 Dilution I = 0.334% alkaloids and Variant 3 - Dilution II = 0.083% alkaloids.
In the extract of Festuca rubra: Variant 1 - Concentrate = 11.60% alkaloids; Variant 2 Dilution I = 5.80% alkaloids and Variant 3 - Dilution II = 1.45% alkaloids.
In the extract of Dactylis glomerata: Variant 1 - Concentrate = 0.053% alkaloids, Variant
2 - Dilution I = 0.026% alkaloids and Variant 3 - Dilution II = 0.006% alkaloids.
In the extract of Poa pratensis: Variant 1 - Concentrate = 0.080% alkaloids, Variant 2 Dilution I = alkaloids and Variant 3 - Dilution II = 0.010% alkaloids.
The results showed the following:
At the seeds of Lolium perenne, the germination at the control registered 92.33%. The
application of some concentrated doses of extracts (variant 1) had a totally inhibitory effect on
the Lolium perenne treated seeds. In variant 2, the inhibitory effect was not so predominant,
ryegrass seed germination in Festuca rubra L. extract was of 39%, 12.33% in the Dactylis
glomerata L. extract and 10.33% in the Poa pratensis L.extract.
In variant 3, the seed germination percentages were the highest, but also lower as in the
case of the untreated control.
At Poa pratensis seeds, variant 1, from all three extracts obtained from the aerial part of
the studied graminaes, has a total inhibitory effect on germination.
In variant 2, there is a total inhibitory effect in the case of Poa pratensis L. seeds treated
with the extract obtained from Festuca rubra L. and Dactylis glomerata L. Dactylis glomerata L.
extract, as compared to the other extracts , has the strongest allelopathic character on Poa
pratensis L., because no matter of the dilution, Poa pratensis L. seeds germination was totally
inhibited.
As the concentration decreases more and more, in variant 3 respectively, Poa pratensis L.
seeds had a germination of 9.67% in the Festuca rubra L. extract and of 29% in the Lolium
perenne L. extract.
Dactylis glomerata L. seeds germination in all three extracts from variant 1 is totally
inhibited. With the dilution of the allelopathic substances, the percentage of seeds germination is
V
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
higher. As in the case of the other extracts, variant 3 with an allelopathic substances
concentration lower than variant 1, has a lower inhibitory effect on Dactylis glomerata L. seeds.
The same phenomenon occurs in the case of Festuca rubra also , with the difference that
the germinated seeds percentage differ according to the applied extract and used dose.
The obtained experimental results show that the extracts have an inhibitory allelopathic
activity on seeds germination and the application of doses of extracts in different concentrations
show that as the allelopathic substances concentration increases, the inhibitory effect is also
increased.
The plants’ growth was also influenced by the applied extracts so that plants kept in an
environment treated with extracts had the coleoptiles and root smaller as compared to the
control, the effect is maximum at the highest concentration.
Statistical processing showed that the length of the coleoptile and root at all four species,
when kept on a environment treated with extracts, were significantly smaller as compared to the
untreated control.
Based on the results, our researches highlighted the fact that the species used to obtain
extracts have had in their composition alkaloids that were identified as having an inhibitory
effect on the growth of the perennial graminaes.
The inhibition of plant growth with the allelopathic substances shows that these
substances also bring competitiveness and, at the same time, superiority to the allelopathic
specie, in relation to the other species.
CHAPTER 6. Results on the influence of extracts on the growth of perennial graminaes
In this chapter we aimed at studying the effect of several extracts from perennial
graminaes on plants growth and development.
The experiment has been installed in three repetitions. Excepting the control, plants from
the other vegetation pots were treated with low-alcoholic extracts of pyrrolizidine alkaloids
respectively ergotics obtained from the aerial part of the studied plants. The extracts were
applied in three different variants:
VI
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
Variant 1 - dose of 10 ml/pot;
Variant 2 - dose of 40 ml/pot;
Variant 3 - dose of 80 ml/pot.
Regarding the influence of the extracts containing alkaloids on perennial ryegrass growth
and development, our experiment has shown that as the quantity and concentration of alkaloids
increases, Lolium perenne growth and development is negatively affected.
In variant 1, shoot length at Lolium perenne has a significantly higher increase when
treated with Festuca rubra extract and distinctly significant higher when treated with Poa
pratensis extract.
If the dose is increased, meaning 40 ml/Pot (variant 2), shoot length at Lolium perenne
treated with Festuca rubra extract is the same as at the first dose meaning significantly higher,
same results were obtained in the case of the perennial ryegrass when treated with Poa pratensis
extract.
If at lower doses,
respectively in variant 1 and variant 2,
there is an increase of the
shoot length at treated Lolium perenne, at a higher dose, namely in variant 3, shoot length is
negatively affected in that it is significantly lower than the control .
The number of leaves at Lolium perenne is also influenced by the applied dose. Applied
doses of extract in variant 1 and variant 2 do not negatively influence this number, but applying a
higher dose of extract (variant 3) leads to the decrease of the number of leaves, this being
significant or distinctly significant lower than the control.
The number of shoots is also negatively influenced when Lolium perenne was treated
with extracts in variant 3, which is very significant or distinctly significant lower than the
control.
By comparing treated Lolium perenne with treated Dactylis glomerata, the same thing
can be observed, the length of the shoot, the number of leaves and the number of shoots are
negatively affected as the dose of extract is increased, so as lolinic alkaloids quantity in the
extracts is higher.
Poa pratensis and Festuca rubra, as well as the other two species, are also influenced by
extracts containing lolinic alkaloids.
VII
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
If at low doses of extracts containing lolinic alkaloids a stimulation of the treated plants is
observed, at higher doses, in our case 80 ml/pot, a inhibition of plant development is observed.
All four species of treated perennial graminaes have intolerance to high doses of extract.
CHAPTER 7. Experimental results on morphological characters exteriorization
The study of the morphological characters was performed separately on each specie
(Dactylis glomerata L., Lolium perenne L., Festuca rubra L., Poa pratensis L.). They were sown
in mixture at three different distances between rows (4 cm, 8 cm, 12.5 cm), the studied specie
sown in pure culture being used as a control.
The studied morphological characters were: height of the bush, the number of shoots in a
bush and the number of leaves on the shoot.
• In the first year of vegetation the height of the bushes at Lolium perenne sown in
mixture at 8 cm distance between rows is smaller than the control variant. As the distance
between rows increases, the height of the Dactylis glomerata bushes sown in mixture is
smaller. A significantly negative difference of Festuca rubra bushes’ height is present
only in the variant sown at 8 cm between rows. Poa pratensis bushes’ height is smaller
than in pure culture only at a distance of 8 cm between rows.
• The number of Lolium perenne shoots is higher than in pure culture in all variants.
Dactylis glomerata sown in mixture with Poa pratensis is the only variant that has a
smaller number of shoots than the control variant. Festuca rubra has a higher number of
shoots than the control variant when sown in mixture with Poa pratensis at 8 cm between
rows and with Dactylis glomerata at 12.5 cm distance between rows. Poa pratensis sown
at 4 cm between rows and in mixture with Lolium perenne and Dactylis glomerata has a
number of shoots significantly higher than the control.
• The number Lolium perenne leaves is different from the control only in the variant of
mixture with Festuca rubra at 12.5 cm between rows and Poa pratensis at 4 cm between
rows. Dactylis glomerata doesn’t show a significant difference of the leaves number.
Festuca rubra has big a number of leaves only in the variant sown in mixture at a
VIII
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
distance of 8 cm between rows. The Poa pratensis leaves number is higher only in the
variant sown at 4 cm between rows.
• In the second year of vegetation, in terms of bushes’ height, Poa pratensis is
negatively affected when sown in mixture with the other studied graminaes. The Festuca
rubra bushes’ height when sown in mixture is smaller than the control variant with one
exception when it is sown with Poa pratensis at 4 cm distance between rows. Dactylis
glomerata has a greater bush height when in mixture with other graminaes at a distance
between rows of 4 cm and when in mixture with Lolium perenne at 8 cm respectively
12.5 cm between rows. As compared to the pure culture, Lolium perenne has a higher
bush height only in mixture with Poa pratensis at a distance of 8 cm and 12.5 cm
between rows.
• The number of Lolium perenne shoots is greater at a distance between rows of 8 cm and
4 cm only with Dactylis glomerata. Dactylis glomerata has a greater number of shoots as
compared to the control, when sown with Poa pratensis at 4 and 8 cm distance between
rows and with Festuca rubra at 12.5 cm. Festuca rubra is negatively affected in terms of
shoots’ number, regardless of the distance between rows and the mixture. There is only
one exception when Festuca rubra is sown in mixture with Poa pratensis at a distance
between rows of 8 cm. At Poa pratensis the number of the shoots increases with the
distance between rows.
• The number of leaves at Festuca rubra is generally greater or the same when sown in
mixture with the other graminaes, the same happens in the case of Poa pratensis. Dactylis
glomerata has a greater number of leaves at a distance between rows of 12.5 cm in
mixture with Poa and Lolium; Dactylis glomerata is otherwise negatively affected by the
other graminaes. Lolium perenne is negatively influenced by Dactylis glomerata in what
concerns the number of leaves on the shoot when is sown at 8 respectively12.5 cm.
• In the third year of vegetation, Lolium perenne bush height is positively influenced only
when is sown with Poa pratensis. Dactylis glomerata has a smaller bush height as
compared to the control when sown in mixture at a distance between rows of 4 and 8 cm.
Festuca rubra and Poa pratensis have a higher bush height when sown with Dactylis
IX
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
glomerata. Festuca rubra and Poa pratensis have a higher bush height at 12.5 cm when
sown with Lolium perenne.
• The number of Lolium perenne shoots is smaller only in mixture with Poa and Festuca
at 12.5 cm distance between rows, in the other variants the number of shoots is equal or
greater than the witness. Festuca rubra is negatively affected by the other species
regardless of the distance between rows with two exceptions when is sown with Lolium
perenne at 4 cm and with Dactylis glomerata at 12.5 cm. The number of Poa pratensis
shoots increases with the increasing of the distance between rows, only when sown with
Lolium perenne, Poa pratensis has a smaller number of shoots. Dactylis glomerata has a
smaller or equal number of shoots as compared to the control, when sown in mixture. It
has a greater number of shoots only in mixture with Poa at 4 cm and Festuca at 12.5 cm.
• Lolium perenne has the same number of leaves in all analyzed variants. The number of
leaves at Festuca rubra increases with the increasing of the distance between rows, same
happens in the case of Dactylis glomerata. Poa pratensis has a greater number of leaves
as compared to the pure culture when sown at 4 cm distance between rows and in mixture
with Dactylis glomerata at 12.5 cm between rows; otherwise the number of leaves is
smaller.
CHAPTER 8. The analysis of the vegetal extracts and their action on the chemical
composition of the plants
Given the complexity of the mechanisms by which perennial graminaes interact, we
considered in this chapter the hypothesis of the allelopathic properties at the four perennial
graminaes in laboratory conditions, from the point of view of the type of chemical compounds,
secondary metabolites.
In this paper, the polyphenolic compounds from the chemical composition of studied
plants were identified and quantified; alkaloids, secondary metabolites that naturally occurred as
a result of interactions between plant and soil were determined.
Polyphenols were determined because the default working protocol in determining
allelopathic substances (NAL - N acetylloline, NFL - N formylloline and EGV - ergovaline)
X
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
requires as extraction solvents the mixture between ethylic alcohol and water at a rate of 20% or
1 5. That fact determined that in the crude vegetal extracts, polyphenols were present.
Polyphenols are synergists with EGV and lolines, thus they serve as catalysts amplifying
the allelopathic effect.
The content of total phenolic compounds in the four studied plant types varies greatly
from one specie to another.
HPLC (high pressure liquid chromatography) analysis of the extracts corresponding to
the four species of graminaes have shown 6 compounds from the phenolic acids class: PCA- pcumaric acid, FA-ferulic acid, , CA- caffeic acid, PHB- p-hydroxybenzoic acid, VA-vanilic acid
and SA- siringic acid. Five of them appear in all extracts and the last (caffeic acid) only in
Dactylis glomerata and Poa pratensis extracts. Three of these products are derivatives of
cinnamic acid (p-cumaric, ferulic respectively caffeic acid), and three are derivatives of benzoic
acid (p-hydroxybenzoic, vanilic, siringic acids).
The results show that phenolic acids are found in studied plants both in linked form and
in free condition.
The analysis of the results highlights the fact that the linked forms of the phenolic acids
are much greater than the free forms. Thus if we take the example of Festuca rubra extract it is
observed that the sum of free form phenolic acids concentrations is 793 ppm (parts per million)
while the sum of linked form phenolic acids concentrations is 10809 ppm. The same trend is
observed in all cases excepting for example the case of Dactylis glomerata extract respectively
the Lolium perenne extract where the differences are small (328 ppm to 1998 ppm).
Chemical studies conducted on perennial graminaes and not only, have showed that the
substances that are causing the apparition of allelopathy are secondary metabolites of vegetal
origin that are a part of the classes of alkaloids, isoprenoids, flavonoids, phenols, terpenoids and
glucosinolates. Unlike the primary metabolites (several hundreds of compounds with low
molecular weight), tens of thousands of compounds with a secondary role are known today, but
only a limited number of them can be categorized as allelochemical substances. These substances
are virtually present in all plant tissues, but in practice they are concentrated in leaves, roots and
vegetal remaining after plant death.
XI
Teză de Doctorat
Aspecte alelopatice la gramineele perene
Biolog BOSTAN Cristian
_____________________________________________________________________________________________
Taking as reference specialty data, we have made the determination of the ergovaline
respectively lolinic alkaloids content in the studied vegetal extracts in order to see the quantity in
which they are found.
Lolinici alkaloids represented by NAL - N-acetylloline, NFL - N-formylloliner and EGV
– ergovaline are found in high concentrations in Lolium perenne respectively Festuca rubra
extracts and less in Dactylis glomerata respectively Poa pretensis extracts.
In Festuca rubra extracts the N-formylloline concentration is 1550 [μg/g], the
concentration of N-acetylloline is 771 [μg/g] and ergovaline has a concentration of 0.149 [μg/g].
The lolinic alkaloids concentration in Lolium perenne extract was as follows:
- NFL - 541 [μg/g]
- NAL - 127 [μg/g]
- EGV - 0.071 [μg/g]
Poa pratensis extract shows the following concentrations of lolinic alkaloids : NFL - 56.7
[μg/g], NAL - 24.12 [μg/g], EGV – undetermined.
In the Festuca rubra extract the concentration of N-formylloline is 42.9 [μg/g], the
concentration of N-acetylloline is 10.67 [μg/g] and ergovaline has not been determined.
The chemical composition of forage plants is one of the main characteristics in terms of
forage quality, the degree of digestibility and consumption depending on the chemical
composition.
One of the major objectives of our researches was to determine the capacity of the
determined substances to influence the quality of the studied graminaes.
Thus, the spraying with extracts leads to changes in the chemical composition of plants,
changes that vary with the applied dose. Therefore the value of the NFL, NAL, EGV
concentrations increases with the increasing of the dose of extract. The application of some doses
of extracts which presented alkaloids in their composition had an influence on quality index,
their influence is reflected by a decrease in crude protein content.
PhD thesis entitled "Allelopathic aspects in the perennial graminaes "
ends with
recommendations and conclusions on the effect of the allelopathic substances on the four species
of perennial graminaes and their allelopathic potential (character).
XII