Research Article Propagation and Introduction of Arnica ...downloads.hindawi.com/journals/tswj/2013/414363.pdfparasite found in mountain arnica ower heads being a limiting factor for

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 414363 11 pageshttpdxdoiorg1011552013414363

Research ArticlePropagation and Introduction of Arnica montana L intoCultivation A Step to Reduce the Pressure on Endangered andHigh-Valued Medicinal Plant Species

Danuta Sugier1 Piotr Sugier2 and Urszula Gawlik-Dziki3

1 Department of Industrial and Medicinal Plants University of Life Sciences in Lublin Akademicka 15 20-950 Lublin Poland2Department of Ecology Faculty of Biology and Biotechnology Maria Curie-Skłodowska UniversityAkademicka 19 20-033 Lublin Poland

3Department of Biochemistry and Food Chemistry University of Life Sciences Skromna 8 20-704 Lublin Poland

Correspondence should be addressed to Urszula Gawlik-Dziki urszulagawlikuplublinpl

Received 8 August 2013 Accepted 2 September 2013

Academic Editors T Hatano C Lu and H Ren

Copyright copy 2013 Danuta Sugier et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Arnica montana (L) is an endangered and endemic medicinal plant species in EuropeThe pressure on natural sources of this plantis alleviated by a suitable use of arnica resources in the European region and introduction into cultivation The objective of thisstudy was to describe the impact of different ways of plant propagation and introduction on the growth and reproduction modeof this species During the six consecutive years of the field experiment the vegetative and reproductive traits were monitoredand survival time was assessed The particular ways of arnica plant propagation and introduction determined all the intrinsicspecies traits and plant survival The values of the characteristics studied indicated good acclimatization of the arnica ecotype tothe climatic conditions of eastern Poland Practical implications from the data presented here include the possibility of using thepresented modes of arnica propagation and introduction in the short- and long-term perspective of arnica cultivation which cangive a possibility of better adjustment of raw material production

1 Introduction

Arnica montana L (Asteraceae) is a rhizomatous herbaceousperennial herb and a medicinal plant widely used as a herbalremedy occurring throughout the entire range of the speciesfrom South Norway and Latvia southwards to the Apenninesand the South Carpathians [1] This species prefers acid andpoor soil conditions It is mainly found in grasslands andshrublands and alpine mountain environments A montanaalso grows in dry pine forests meadows on siliceous soilsmarginal parts of spruce forests open forest edges mowingpastures and margins of peatlands [2ndash7] The species is acomponent of the five habitat types of the Habitats Directive[8]

Eutrophication habitat fragmentation and agriculturalintensification have led to a rapid decline in the mountainarnica in many European countries [9 10] A montanahabitats have been fragmented especially at the edge of its

dense geographical range [11] The negative influence on thearnica population comes from such competitors as Dactylisglomerata [12] Agrostis capillaris [7] Deschampsia flexuosaand Calamagrostis villosa [10] Additionally the main seriousdamage is caused by slug herbivores and the specialist flyTephritis arnicaemdashhighly specialized onAmontana the onlyparasite found in mountain arnica flower heads being alimiting factor for the geographical and mountain altituderange of this species [13 14]

Arnica montana is a medicinally important plant specieswidely applied in pharmacy homeopathy and cosmetics Dif-ferent plant parts such as inflorescences rhizomes roots andleaves are collected for healing purposes Arnica is a sourceof sesquiterpenes essential oils terpenoids sesquiterpenelactones flavonoids and phenolic acids especially chloro-genic acids [15ndash17] and exhibits antiseptic anti-inflammatoryantibacterial antisclerotic antifungal and antioxidant activ-ities [15 18] Recently new derivatives of chlorogenic acids

2 The Scientific World Journal

in A montana flowers and the composition of essential oilsin rhizomes and roots have been identified and characterized[19 20]

A montana is a rare plant under strict protection andis included in the IUCN Red List of Threatened Species [8]and in the Red Data Books and Red Data Lists of manyEuropean countries [21 22] Despite the loss of habitats Amontana is mainly harvested from the wild Dried flowerstraded annually in Europe are estimated to be around 50 t[23]The collection ofAmontana formedicinal purposes hasalso caused disappearance or reduction in the size of severalEuropean populations [24] The pressure on natural sourcesof this plant is alleviated by a suitable use of arnica supplyin the European region where flower heads are harvested[25] A montana is protected by law however this form ofprotection is insufficient Therefore over the last decadesother forms of active protection have appeared such asrestoration andmanagement of its natural habitats [9 26 27]plant introduction [27 28] and attempts at field cultivation[20 29 30] Biotechnological approaches for cultivation andenhancement of secondary metabolites in these species arevery interesting [31 32]

The objective of this study was to describe the impactof different ways of plant propagation and introduction onthe growth and reproduction mode of this species in thearea of its geographical range in the eastern part of Polandand to obtain information on their suitability for climaticand edaphic conditions During the six consecutive years ofthe experiment the vegetative and reproductive traits weremonitored as potentially important factors influencing theplant establishment success [33] In the future the knowledgeof the part of the life history of these species can be helpfulin cultivation which may reduce the pressure on naturalpopulation and reintroduction of A montana into theirnatural stands where the abundance of arnica has declined

2 Materials and Methods

21 Experimental Site Conditions In the present study weused individuals taken from Botanical Garden UMCS inLublin The 6-year-long experiments (2007ndash2012) were per-formed on experimental fields at the University of LifeSciences in Lublin located in the eastern part of Poland51∘331015840N 22∘441015840 E on grey-brown podsolic soil with thegranulometric composition of heavy loamy sand This sitewas chosen because the soil has physicochemical propertiescomparable to natural soil conditions for arnica [7 9]

The climate of the Lubelszczyzna region ismainly affectedby penetrating polar-oceanic masses of air and polar massesof continental air The growing season in the middle andeastern part of these region lasts 211ndash214 days [34] Theaverage month precipitation ranged from 10mm in Novem-ber 2011 to 189mm in July 2011 and the average monthtemperature ranged from minus81∘C in January 2010 to 216∘C inthe cultivation years 2007ndash2012 (Figure 1) However themeanannual sum of precipitation in the study period ranged from5102mm in 2012 to 7788mm in 2010 and the mean annualtemperature ranged from 798∘C in 2010 to 959∘C in 2008

In order to determine the edaphic conditions beforeplantation establishment topsoil samples were randomlyselected from the depth of 15 cm The main soil proper-ties were analysed The topsoil was characterized by anaverage content of organic matter (155) high phospho-rus (841mg Psdotkgminus1 PN-R-04023 1996) average potassium(722mg Ksdotkgminus1 PN-R-04022 1996+Az12002) very lowmagnesium (24mg Mgsdotkgminus1 PN-R-04020 1994+Az12004)and acid reaction (pH inH

2Omdash418 and in 1molsdotdmminus3 KClmdash

379 PN-ISO 10390 1997)

22 PlantMaterial and Experimental Design Thefield exper-iment was established in April 2007 in three ways of plantestablishment The plants were introduced as clone seedlings(CS) and rosettes (RS) and by sowing (SS)

The randomly chosen ramets taken from edge of 6-year-old genets after their natural disintegration just beforeplanting were transplanted individually into the field Theplants are marked as CS

The nine-month-old rosettes that were established afterspontaneous seed germination and originated from the col-lection subsequently they were individually transplantedinto small pits The plants are marked as RS In anotherfield experiment conducted in recent years to assess themortality rates of plants spontaneous seedling recruitmentwas observed hence the idea of using thereof for introductioninto cultivation

A montana seeds (achenes) for sowing were collectedfrom 15 individuals from the arnica collection at the endof June 2006 and stored dry and at room temperature Atthe end of April 2007 only dark and firm achenes wereselected and sown into rows where soil taken from motherplantation was scattered Our earlier experiments indicatedthat soil taken from plants compared to commonly usedstandard substrates exerted a significant impact on seedlingemergence and plant growth After sowing the soil wasslightly pressed to improve the soil contact with the seedsTen weeks after sowing when the seedlings formed rosettescharacterized by 3-4 leafs individuals were left in rows at20 cm intervals The plants are marked as SS

The A montana seeds were sown and the seedlings weretransplanted at a 40 times 20 cm distance thus ensuring almostthe same plant densities (125 plantsmminus2) The experimentalunit was made up of 75 plants The experimental design wasa randomized complete block with four replications (plotsof 6mminus2) During the vegetation period the plantation wasthree times weeded by hand We observed vegetative andreproductive traits and survival of genets from the pointof view of species biology therefore the flower heads werenot harvested Simultaneously the same characteristics werestudied in the other field experiment after harvesting flowerheads The comparable genets from the experiments withand without head harvest did not differ statistically hencethe harvest of flower heads did not determine the traitsstudied

We present changes in the characteristics studied cal-culated per genet containing several ramets formed duringthe vegetative growth In the last year of observation we

The Scientific World Journal 3

Time (years)2007 2008 2009 2010 2011 2012

020406080

100120140160180200

Mon

thly

pre

cipi

tatio

n su

m (m

m)

PrecipitationTemperature

0510152025

minus5

minus10

Aver

age m

onth

ly te

mpe

ratu

res (

∘

C)

Figure 1 Monthly average temperature and monthly precipitation sum at studied region for six successive study years 2007ndash2012

Table 1 The effects of three different ways of introduction into cultivation on the studied traits of A montana genets (ANOVA Kruskall-Wallis test) The 119865 and 119867 values are shown The levels of significance are indicated by an asterisk (lowast001 lt 119875 le 005 lowastlowast0001 lt 119875 le 001lowastlowastlowast

119875 le 0001) Explanation see Section 23

2007 2008 2009 2010 2011 2012TNR119865 4024lowastlowastlowast 5048lowastlowastlowast 3417lowastlowastlowast 1901lowastlowastlowast 17890lowastlowast 25602lowastlowast

NFR119865 mdash 17507lowastlowast 1527lowastlowastlowast 4704lowastlowastlowast 18719lowastlowast 38272lowastlowast

HS119867 mdash 6739lowastlowastlowast 6739lowastlowastlowast 6739lowastlowastlowast 6739lowastlowastlowast 6739lowastlowastlowast

NH119867 mdash 2105lowastlowastlowast 4309lowastlowast 4652lowastlowastlowast 6498lowastlowastlowast 6659lowastlowastlowast

NHFS119865 mdash 1559lowastlowastlowast 364lowast 133522lowastlowastlowast 2406lowastlowastlowast 4382lowastlowastlowast

AH119867 mdash 2105lowastlowastlowast 4309lowastlowastlowast 4652lowastlowastlowast 6498lowastlowastlowast 6659lowastlowastlowast

CAH119867 mdash 6244lowastlowastlowast 2754lowastlowastlowast 5988lowastlowastlowast 6540lowastlowastlowast 7214lowastlowastlowast

CNH119865 mdash 38997lowastlowast 13275lowastlowast 1845lowastlowastlowast 17939lowastlowast 57311lowastlowast

Survival119867 65 561 768lowast 1489lowastlowastlowast 1377lowastlowastlowast 1532lowastlowastlowast

observed disintegration of genets and formation of cloneclusters Despite this fact we defined the units as genets thatis genetic individuals containing some ramets

23 Measurements under Field Conditions Each summerat the peak of flowering 30 individuals per each mode ofplant establishment were measured when available Severalvegetative and reproductive characters were measured thetotal number of ramets per genet (TNR) the number offlowering ramets per genet (NFR) the height of floweringstems per genet (HS) the number of heads per genet(NH) the number of heads per flowering stem of the genet(NHFS) and the head diameter per genet The total areaof heads per genet (AH) was calculated on the basis ofthe head diameter On the basis of 6-year observation thecumulative area of heads per genet (CAH) and the cumulativenumber of heads per genet (CNH) were determined Themorphological and reproductive parameters were recordedfrom 30 plantsrepetition

The survival time was assessed by recording survivalof individual plants each June from the beginning of theexperiment until July 2012 A plantwas considered deadwhenit was not found during subsequent censuses The presentedclimatic data were obtained from the Felin climatic station

24 Statistical Analysis Prior to the analysis all data weretested for normality with the Shapiro-Wilk test Varianceheterogeneity was checked using a Levenersquos test Parametricdatawere tested using one-way analysis of variance (ANOVA)with subsequent Tukey tests (data were normally distributedwith homogeneous variances) When the data were notnormally distributed or the variance was not homogeneousthe differences between the 3 modes of introduction weretested using Kruskal-Wallis tests with subsequent Mann-Whitney 119880-tests All results are expressed as means plusmn SDand the differences were considered significant 119875 lt 005All statistical analyses were carried out using the Statisticasoftware programme

3 Results

The total number of ramets per genet (TNR) differed signif-icantly between the three different modes of plant establish-ment in each study year (Table 1 Figure 2) In the first fullvegetation season (2007) plants introduced as CS formed 2ndash6 new offspring ramets (mean 54) and genets introduced asRS formed 3ndash8 new offspring ramets (mean 64) Howeverindividuals grown from seeds produced 2ndash5 new offspringrosettes (mean 34) In the second year of observation theCS genets had a higher TNR while in the third year the RS


CSRSSS

0

15

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3025

35

10

2007 2008 2009 2010 20122011

a

a

c

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a

a

bb

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Time (years)

Figure 2 The total number of ramets per genet (TNR) in different ways of A montana introduction into cultivation The plants wereintroduced as clone seedlings (CS) and rosettes (RS) and by sowing (SS) Bars (means) followed by the different letters within the samestudy year differ significantly (Tukey-test 119875 lt 005)

05

1510

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40

3025

35

cb

a

c

ba

ba

cb

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acb

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CSRSSS

2007 2008 2009 2010 20122011Time (years)

Figure 3 Number of flowering ramets per genet (NFR) in different ways of A montana introduction into cultivation Explanation seeFigure 2

were characterised by a higher TNR In the fourth fifth andsixth study years the CS genets had a significant lower TNRper genet than those of the RS and SS however there was nosignificant difference between the RS and SS

In the first year of observation the genets introduced asRS and SS did not generate flowering stems However themean number of flowering stems produced by individualsgrown from clones was 18 In each subsequent study yearthere was a significant difference in the total number offlowering ramets per genet (NFR) between the threemodes ofestablishment (Table 1 Figure 3) In 2008 the NFRs in the CSwere higher than those in the RS and SS However in 2009the NFRS in the RS were higher than those in both the CSand SS genets In 2010ndash2012 the number of flowering rametswas significantly higher in the SS plants in comparison to CSand RS In 2011 the NFR in the CS plants was over two timeslower than in both RS and SS and ca 7 times lower in 2012respectively

In each study year there was a significant difference inthe height of the flowering stems per genet (HS) betweenthe three modes of plant establishment (Table 1 Figure 4) In

2008ndash2010 the HS in both the RS and SS genets was higherthan in the CS plants In the last two years of observation theHS in the SS individuals was significantly higher than in theCS and RS genets

The number of heads per genet (NH) differed signifi-cantly between the three different modes of plant establish-ment in the all study years (Table 1 Figure 5) In the secondvegetation season (2008) plants introduced as CS produceda mean of 49 heads and only in that year their numberwas significantly higher in relation to genets introduced asRS and SS In 2009 the NH of both RS and SS genets wassignificantly higher than in the CS individuals In the nextyears of the experiments the NH in plants introduced asseeds was significantly higher in relation to the RS and CSgenets

The number of heads per flowering stem per genet(NHFS) differed significantly between the three differentmodes of plant establishment excluding the fourth (2010)year of observation (Table 1 Figure 6) The highest NHFS inthe range of 4ndash8 was produced by RS genets in the secondgrowing season and the mean value of this parameter was


0

30

10

40

80

6050

70

20

a ab bb b a

b b

a abb c c

CSRSSS

2007 2008 2009 2010 20122011Time (years)

Figure 4 The height (cm) of flowering stems per genet (HS) at different ways of A montana introduction The plants were introduced asclone seedlings (CS) and rosettes (RS) and by sowing (SS) Bars (means) followed by the different letters within the same of study year differsignificantly (Mann-Whitney 119880-test 119875 lt 005)

0

40

20

60

80

100

120

a

bc

a bba

bc

c cb b

aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)

Figure 5 The number of heads per genet (NH) in different ways of A montana introduction Explanation see Figure 4

01

32

4

7

56 ab

b

a ba

bab

a

cb

a

a aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)

Figure 6 The number of heads per flowering stem of the genet (NHFS) in different ways of A montana introduction Explanation seeFigure 2

higher than in the CS and SS genets In the third year ofvegetation theNHFS in the CS genets was comparable to thatin the SS plants and significantly higher in relation to the RSgenets However in the last two years of the experiment theNHFS in the SS plants was higher than that in the RS and CSgenets

The area of heads per genet (AH) differed significantlybetween the three different modes of plant establishmentin all study years (Table 1 Figure 7) In the second yearof observation the AH of genets introduced as CS and RSwas comparable and was over three times higher in relationto plants introduced as seeds In each consecutive year


0

45004000350030002500200015001000

500

b

a

ba

ab

a

cb

a

b

cb

c

c

CSRSSS

2007 2008 2009 2010 20122011Time (years)

Figure 7 The area of heads (cmminus2) per genet (AH) in different ways of A montana introduction Explanation see Figure 4

002

0604

08

14

1012

ba

b aa

b

a

cb

ab

c

c

a

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)

Figure 8 The cumulative area of heads (mminus2) per genet (CAH) in different ways of A montana introduction Explanation see Figure 4

the highest area of heads was characteristic for individualsintroduced as seeds In 2009ndash2012 the AH in both the SS andRS plants was higher than in the CS genets respectively

The cumulative area of heads per genet (CAH) differedsignificantly between the three different modes of plantestablishment in all study years (Table 1 Figure 8) The CAHin genets introduced as CS was higher than that in the caseof the other ways of introduction only in the second year ofobservation (2008) In the third growing season the CAHin the RS plants was higher than in both the CS and SSgenets In the fourth study year theCAH in plants introducedas rosettes and by sowing was comparable as well as inrelation to the CS plants However in the last two years ofthe experiment the CAH in plants introduced as seeds washigher than in individuals introduced as rosettes and cloneseedlings

There was a significant difference in the cumulativenumber of heads per genet (CNH) between the three modesof introduction (Table 1 Figure 9) In the second and thirdgrowing seasons the CNH of genets introduced as CS washigher than in genets introduced as RS and SS and inthe next years of the experiment it was significantly lowerrespectively In the fourth study year the CNH was thehighest in RS plants and in the fifth study year the CNHwas comparable in the RS and SS genets However in the last

year of observation the CNH in the SS individuals wassignificantly higher than in the RS and CS plants

In the first two years after plant establishment in thedifferent modes plant survival was comparable howeverin the consecutive years the survival differed significantlybetween the three different modes of plant introduction(Table 1 Figure 10) Nearly six years after introduction thesurvival of genets introduced as CS was over 3 times lowerthan in RS plants and over 4 times lower compared toindividuals introduced as seeds

4 Discussion

41 Propagation and Introduction of A montana In thedifferent cultivation experiments the preferable method ofestablishment for large-scale planting is to raise plants fromseeds under glasshouse conditions and transplant them intothe field [29 30 35 36] In our studies besides sowing inthe field spontaneously established seedlings (rosettes) takenfrom the arnica collection and edge clones taken from 6-year-old disintegrated genets were used The ramets and cloneswere transplanted directly to the ground without the seedlingpreparation stage which can evoke stress in young plantsSimultaneously it is necessary to underline the reduction of


050

150100

200

350300250

abc

a

ac b

b c abb

a

bc

CSRSSS

2007 2008 2009 2010 20122011Time (years)

Figure 9 The cumulative number of heads per genet (CNH) in different ways of A montana introduction Explanation see Figure 2

0

20

60

40

80

100 aa ba

ab

a

c

b

a

aaa ab cba

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)

Figure 10 The survival () of A montana individuals in different ways of introduction Explanation see Figure 4

time designed to preparation of seedlings and economicalaspect

One of the ways of plant introduction in the presentedstudies was using clone seedlings In studies on arnicaintroduction on Tara mountain in Serbia in the variant ofspring planting with clonal seedlings taken by dividing 2-year-old genets the seedlings were almost completely lost[20] In our studies we used clonal seedlings taken from6-year-old clusters already after genet disintegration nextthe particular ramets were naturally divided and containedparts of rhizomes and roots Therefore in the first year of theexperiment the plants formed new ramets and flowered incontrast to the plants established in the other two modes

In A montana cultivation plant survival after sowing orseedling introduction is rarely reported In an experimentof arnica introduction through seedlings derived from seedson an abandoned arable field where the top layer had beenremoved to reduce competition the survival in the first yearafter plant establishment was about 50 [28] whereas in ourstudies it exceeded 90Our data are in good agreementwiththe results of an experiment on arnica conducted in a naturalheath area [27] and are also in agreement with the experimentconducted under controlled greenhouse conditions wherea 90 survival rate was observed when seedlings wereinoculated with mycorrhizal fungi [12] On the contrary

in that experiment the nonmycorrhizal plants were severaltimes smaller and were dark green and their survival ratestrongly decreased with time going down to about 16 at theend of the one-year cultivation period

In the present study we focused on introduction ofplants using seedlings spontaneously established from ach-enes During the 6-year study period rapid growth andreproduction and very low mortality of genets especially inthe first three years of the experiment were observed Thismode of plant introduction is more likely to eliminate poorlyadapted individuals Moreover the use of clone seedlingsrosettes and soil from A montana collection before sowingprobably guarantees the presence of mycornhiza which isalways affirmed in arnica natural habitats [37 38] Thisquestion should be however explained and confirmed infurther studies

42 Vegetative andReproductive Traits In other experimentsthe vegetative and generative traits of cultivated plants weredifferent and dependent on the mode of plant introduction[20 30] This was usually connected with the different ageof cultivated plants especially in the first and second yearsof plant life The results of our studies are in agreement withthese data and indicate that all the modes of plant establish-ment determined both the vegetative and reproductive traits


In the case of genets introduced as rosettes and by sowingthe values of the characteristics studied were increased to thefourth year of cultivation and decreased in the next two yearsThis pattern was in accordance with reports from Finland[35] but is somewhat different from the results of 5-yearobservation in Serbia [29] where the maximum flower yieldwas noted in the third year of cultivation

The number of rosettes per genet reflects clonal growthof plants and is very often counted as a yield component [20]The values of this parameter in plants introduced by sowingwere over 2 times higher compared to data presented fromthe Alps in plants established in the same way [30] Howeverthe number of rosettes per genet of 2-year-old and 3-year-oldplants was slightly lower in relation to data presented fromTara mountain in Serbia [20]

In natural habitats the proportion of flowering arnicarosettes decreasedwith altitude indicating a shift from sexualreproduction to clonal growth and was different in genetswhich are components of different plant communities [6]The number of flowering stems per plant was variable anddepended mainly on their age On average 2-year-old arnicaplant established by sowing formed ca 5 flowering stemswhich was almost 2-fold higher than the results reported byAiello et al [30] In the natural population not all arnicaclusters or rosettes produced generative rosettes and notall individual rosettes within a flowering cluster produceda flowering shoot [2] Similarly in the field or glasshouseconditions plants can produce flowering stems in the nextvegetation season after introduction but the flowering ischangeable [13 28 30 39] However in the present experi-ment the plants introduced as rosettes which were formed inthe previous vegetation season did not flower but produceda high number of ramets The absence of flower heads ingenets introduced as seedlings after a cold winter period witha short day which stimulates flower development [28] canbe explained by the fact that A montana is a clonal plant thatcan allocate resources either to sexual reproduction or clonalgrowth that is production of more rosettes inside genets [6]In favourable field conditions when plant competition wasexcluded arnica genets allocated resources probably in thevegetative growth resulting in a higher number of ramets andlack of flowering at 2-year-old plants

The total area of flower heads per genet is strictly relatedto the flower head diameter and reflects the flower head yield[18] Moreover the number of seeds per flower head is highlycorrelated with the flower head diameter and length of theflower stem [4] In the case of genets established in all thethree modes the value of this parameter was the highest inthe fourth year of cultivation Similarly the number of flowerheads per plant reflects the flower heads yield and in manycases is dependent on the location of the population andsite conditions [40] The values of this parameter in plantsintroduced by sowing were over 2 times higher comparedto the data reported from the Alps in 2-year-old plantsestablished in the same way [30] The maximum number offlower heads in the fourth year of cultivation did not exceed100 whereas many highly productive clones selected fromwild plant populationswere characterized by 134 flower headsper plant [40]

43 Implication for Cultivation From the point of view ofherb material production all the presented three ways ofintroduction can be applied In the first and second vegetationseasons arnica plants introduced as ramets taken from 6-year-old plants after genet defragmentation produced thehighest number of flower heads and exhibited the highesttotal area of flower heads Moreover the plants generatedthe highest number of ramets per genet and the greatestnumber of flowering stems Therefore when collection ofarnica flowers is planned for 2 or 3 successive years theplantation should be established by introduction of cloneseedlings This mode of introduction guarantees high cropsof Arnicae Flos already in the first two years of cultivation

The number of stems per plant and the number of flowersper plant in 2-year-old genets introduced in this way wereover 2 times higher in relation to plants cultivated in Finland[35] introduced as seedlings prepared from seeds and over50 higher in relation to our earlier studies [18]The numberof flowering stems per plant was over two times higher inrelation to plants introduced by sowing in this experimentand almost 2 times higher compared to other results [1835] Therefore if there is an immediate demand for rawmaterial the A montana plantation can be established bytransplantation of clone seedlings taken from many-year-old genets after their disintegration However the very highclonal growth favours production of rhizomes and rootswhich are the main source of essential oils [17 20] Thecorrelation between the rosette number and rhizome yield ishigh [20] In our experiment plants established by ramets hadthe highest number of rosettes per genet however this way ofplant establishment can be used for arnicarhizome materialproduction

In the longer perspective of arnica cultivation and rawmaterial collection the two other modes of introductioncan be considered After 3 vegetative seasons the genetsintroduced by rosettes from the mother plantation are char-acterized by the highest cumulative total area of flower headsper genet However in the perspective of collection of flowerheads in the 3rd or 4th years after introduction establishmentvia rosette taken from the arnica collection or early producedcan be taken into account

The highest yield of flower heads was provided by 3- and4-year-old plants under field cultivation [29 35 36 41] Inthe case of plants introduced as rosettes and by sowing thevalues of all the parameters studied increased to the 4th yearof cultivation and decreased in the next years of observationDespite this fact the cultivation should by continued sincethe total area of flower heads produced by genets in 2011 and2012 years introduced through the firstmodewas comparableto the values noted in 2008 (the second year of cultivation)However the total area of flower heads of the 5- and 6-year-old plants introduced by sowing was only about 30 lower inrelation to the 3- or 4-year-old plants and over 4 times highercompared to the 2-year-old plants

After the 4th vegetative season the cumulative total areaof flower heads per genet in plants introduced as rosettesand by sowing was comparable However in the last twoyears of the experiment the values of the characteristics inplants introduced by sowing were higher in relation to plants


introduced as rosettes Additionally the observed highersurvival of plants established by seeds as compared with theother modes of introduction suggests that sowing is better inthe perspective of 5-6-year arnica cultivation

44 Genet Disintegration Our studies indicate that in veryfavourable field conditions without inter- and intraspeciescompetition (common in natural habitats) A montana canproduce clumping rametswhich are developed from the shortrhizomes of the spreading ramets thereby representing aphalanx growth form [42 43] A montana genets have acompact structure but in the 4th or 5th year of observation(depending on the way of introduction) we noticed necro-mass accumulation and lack of production of new rametsin the central part of the genets In our opinion this isthe first symptom of the beginning of genet division Thedisintegration of genets depended on plant age and is thebeginning of vegetative propagation in many clonal plants[44 45] which had not been described in arnica Aftersix years of the experiment we found that the proportionof disintegrated genets depended on the mode of plantpropagation and was a consequence of genet senescence Allthe genets established as clones were divided In the case ofplants established as rosettes and by sowing the proportionof disintegrated genets was 84 and 45 respectively (datanot presented)

45 Arnica Cultivation under the Climatic and Edaphic Con-ditions The study of the impact of climate conditions on thetraitsstudied over time is difficult and requires many years ofobservation and therefore is rarely undertaken Pljevljakusicet al [20] have presented climatic conditions for three years(2008ndash2010) of A montana cultivation in Serbia but didnot describe the relation between the climatic factors andthe vegetative and reproductive traits In our experimentthe precipitation patterns of the last two experimental yearswere different from the previous years In 2011 the sumof precipitation was over 40 lower in relation to 2009and 2010 and in 2012 it was over 30 lower respectively(Figure 1) Therefore the drastic decrease in the values ofsome studied traits in the case of plants introduced as cloneseedlings in relation to others modes in the last two yearsof the experiment may have been caused by the lowest sumof precipitation especially in the spring months Howeverit should be underlined that the plants whose traits werecompared differed in age which had an impact on thebeginning of genet defragmentation and values of the traitsstudied over time

In nature A montana grows mainly in siliceous moun-tains but it also occurs at lower altitudes [3ndash7 10 11]This species prefers acid and poor soil conditions and isusually dependent on their symbiotic partner [12 38] InPoland arnica grows in lowland areas on acid forest soils(pH 4-45) and in mountain areas on acidophilus subalpinegrassland soils of pH 48-55 [38] In our opinion arnicaplants introduced for cultivation should be cultivated in soilconditions similar to those prevailing in nature Howeverin the case of our studies the plantation was established

on grey-brown podsolic soil with acidic reaction and thegranulometric composition of heavy loamy sandThe topsoilwas characterized by an average content of organic matterhigh phosphorus average potassium and very low magne-sium The physicochemical soil properties are comparable tonatural arnica habitats [7 9 38] Given the growth and Amontana reproduction it can be affirmed that the cultivatedplants have favourable edaphic conditions

5 Conclusion

(1) The particular ways of A montana plant propagationand introduction presented in this work determinedall the intrinsic species traits studied and plant sur-vival

(2) The presented results in relation to another Europeandata indicated that climatic conditions and soil con-ditions are favourable for arnica cultivation in easternPoland

(3) Practical implications from the data presented hereinclude the possibility of using the studied ways ofA montana propagation and introduction in plantcultivation which decreases pressure to natural popu-lations in other European countriesWe suggest usingthe three different modes of arnica propagation andintroduction in the short- and long-term perspectiveof arnica cultivation which can give a possibility ofbetter adjustment of raw material production

(4) From the perspective of multiyear observation of Amontana in the field conditions we can pay attentionto genet disintegration which is an important aspectof the biology of the species The genet division isthe beginning of plant vegetative reproduction and isdetermined as vegetative and reproductive traitsThisphenomenon is very important for the demographydynamics and genetic variation of a natural popula-tion and should be studied in the future

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] E Hulten andM FriesAtlas of North EuropeanVascular PlantsNorth of the Tropic of Cancer vol 1 Koeltz Scientific BooksKonigstein Germany 1986

[2] SH Luijten J G BOostermeijer N C van Leeuwen andHCMDenNijs ldquoReproductive success and clonal genetic structureof the rare Arnica montana (Compositae) in The NetherlandsrdquoPlant Systematics and Evolution vol 201 no 1ndash4 pp 15ndash301996

[3] M Zając ldquoMountain vascular plants in the Polish lowlandsrdquoPolish Botanical Studies vol 11 pp 1ndash92 1996

[4] S Kahmen and P Poschlod ldquoPopulation size plant perfor-mance and genetic variation in the rare plant Arnica montana


L in the Rhon Germanyrdquo Basic and Applied Ecology vol 1 no1 pp 43ndash51 2000

[5] J Mitka T Winnicki and B Zemanek ldquoMountain arnicaArnica montana Lrdquo in Red Data Book of the PolishCarpathiansmdashVascular Plants Vascular Plants Z Mirekand H Piękos-Mirkowa Eds pp 352ndash353 W Szafer Instituteof Botany Polish Academy of Science Krakow Poland 2008(Polish)

[6] T Maurice G Colling S Muller and D Matthies ldquoHabitatcharacteristics stage structure and reproduction of collineand montane populations of the threatened species Arnicamontanardquo Plant Ecology vol 213 no 5 pp 831ndash842 2012

[7] D Wołkowycki ldquoMountain arnica Arnica montana Lrdquo inMonitoring of Plant Species Methodical Guide J PerzanowskaEd part III pp 40ndash51 GIOS Warszawa Poland 2012 (Polish)

[8] A Falniowski I Bazos I Hodalova R Lansdown and APetrova ldquoArnica montanardquo in IUCN Red List of ThreatenedSpecies version 20122 IUCN 2012

[9] M C C de Graaf R Bobbink J G M Roelofs and P J MVerbeek ldquoDifferential effects of ammoniumand nitrate on threeheathland speciesrdquo Plant Ecology vol 135 no 2 pp 185ndash1961998

[10] J Fabiszewski and B Wojtun ldquoContemporary floristic changesin the Karkonosze Mtsrdquo Acta Societatis Botanicorum Poloniaevol 70 no 3 pp 237ndash245 2001

[11] S H Luijten A Dierick J Gerard B Oostermeijer L E LRaijmann and H C M Den Nijs ldquoPopulation size geneticvariation and reproductive success in a rapid declining self-incompatible perennial (Arnica montana) in The NetherlandsrdquoConservation Biology vol 14 no 6 pp 1776ndash1787 2000

[12] A Jurkiewicz P Ryszka T Anielska et al ldquoOptimization ofculture conditions of Arnica montana L effects of mycorrhizalfungi and competing plantsrdquoMycorrhiza vol 20 no 5 pp 293ndash306 2010

[13] U Scheidel S Rohl and H Bruelheide ldquoAltitudinal gradientsof generalist and specialist herbivory on three montane Aster-aceaerdquo Acta Oecologica vol 24 no 5-6 pp 275ndash283 2003

[14] U Scheidel and H Bruelheide ldquoEffects of slug herbivory onthe seedling establishment of two montane Asteraceae speciesrdquoFlora Morphology Distribution Functional Ecology of Plantsvol 200 no 4 pp 309ndash320 2005

[15] M Ganzera C Egger C Zidorn and H Stuppner ldquoQuantita-tive analysis of flavonoids and phenolic acids inArnicamontanaL by micellar electrokinetic capillary chromatographyrdquoAnalyt-ica Chimica Acta vol 614 no 2 pp 196ndash200 2008

[16] U Gawlik-Dziki M Swieca D Sugier and J Cichocka ldquoCom-parison of in vitro lipoxygenase xanthine oxidase inhibitoryand antioxidant activity of Arnica montana and Arnica chamis-sonis tincturesrdquoActa Scientiarum Polonorum HortorumCultusvol 10 no 3 pp 15ndash27 2011

[17] I Weremczuk-Jezyna H Wysokinska and D Kalemba ldquoCon-stituents of the essential oil from hairy roots and plant roots ofArnicamontana Lrdquo Journal of Essential Oil Research vol 23 no1 pp 91ndash97 2011

[18] D Sugier and U Gawlik-Dziki ldquoThe influence of foliar fer-tilization on yielding and quality of mountain arnica (Arnicamontana L) and chamisso arnica (Arnica chamissonis varfoliosa)rdquo Annales UMCS Agricultura vol 64 no 3 pp 129ndash1392009 (Polish)

[19] R Jaiswal and N Kuhnert ldquoIdentification and characterizationof two new derivatives of chlorogenic acids in arnica (Arnica

montana L) flowers by high-performance liquid chromatog-raphytandem mass spectrometryrdquo Journal of Agricultural andFood Chemistry vol 59 no 8 pp 4033ndash4039 2011

[20] D Pljevljakusic D Rancic M Ristic L Vujisic D Radanovicand Z Dajic-Stevanovic ldquoRhizome and root yield of thecultivated Arnica montana L chemical composition and his-tochemical localization of essential oilrdquo Industrial Crops andProducts vol 39 no 1 pp 177ndash189 2012

[21] D Korneck M Schnittler and I Vollmer ldquoRed list of Pterido-phyta and Spermatophyta in Germanyrdquo in Red List of Endan-gered Plants in Germany G Ludwig andM Schnittler Eds vol28 of Schriftenreihe fur Vegetationskunde pp 21ndash187 Bundesamtfur Naturschutz Bonn Germany 1996 (German)

[22] K Zarzycki and Z Szeląg ldquoRed list of the vascular plants inPolandrdquo in Red List of the Plants and Fungi in Poland Z MirekK Zarzycki W Wojewoda and Z Szeląg Eds pp 11ndash20 WSzafer Institute of Botany Polish Academy of Sciences KrakowPoland 2006 (Polish)

[23] T Burfield ldquoUpdated list of threatened aromatic plants used inthe aroma amp cosmetic industriesrdquo 2010 httpwwwcropwatchorg

[24] D Lange Europersquos Medicinal and Aromatic Plants Their UseTrade and Conservation TRAFFIC International CambridgeUK 1998

[25] BMichler ldquoConservation of eastern Europeanmedicinal plantsArnica montana in Romaniardquo Case study Garda de Sus Man-agement Plan 2007

[26] L J L van denBerg P Vergeer and J GM Roelofs ldquoHeathlandrestoration in The Netherlands effects of turf cutting depth ongermination of Arnica montanardquo Applied Vegetation Sciencevol 6 no 2 pp 117ndash124 2003

[27] P Vergeer Introduction of threatened species in a fragmented anddeteriorated landscape [PhD thesis] 2005

[28] S H Luijten M Kery J G B Oostermeijer and H J CM Den Nijs ldquoDemographic consequences of inbreeding andoutbreeding in Arnica montana a field experimentrdquo Journal ofEcology vol 90 no 4 pp 593ndash603 2002

[29] D Radanovic T Markovic S Antic-Mladenovic D Pljevl-jakusic M Ristic and D Krivokuca-Dokic ldquoYield and qualityof Arnica (Arnica montana and Arnica chamissonis var foliosa)cultivated in Serbiardquo in Proceedings of the 1st InternationalScientific Conference on Medicinal Aromatic and Spice PlantsM Haban and P Otepka Eds pp 157ndash161 Nitra Slovakia 2007

[30] N Aiello F Scartezzini and C Vender ldquoCultivation trial ofArnica montana wild accessionsmdashresults of the second yearrdquoActa Horticulturae vol 955 pp 253ndash257 2012

[31] M Petrova E Zayova R Vassilevska-Ivanova andM VlahovaldquoBiotechnological approaches for cultivation and enhancementof secondary metabolites in Arnica montana Lrdquo Acta Physiolo-giae Plantarum vol 34 no 5 pp 1597ndash1606 2012

[32] A Surmacz-Magdziak and D Sugier ldquoIn vitro propagationof Arnica montana L an endangered herbal species of greatimportance to medicinerdquo Acta Scientiarum Polonorum Horto-rum Cultus vol 11 no 2 pp 127ndash140 2012

[33] B R Murray P H Thrall A M Gill and A B Nicotra ldquoHowplant life-history and ecological traits relate to species rarity andcommonness at varying spatial scalesrdquo Austral Ecology vol 27no 3 pp 291ndash310 2002

[34] B M Kaszewski The Climatic Conditions of the Lublin RegionUMCS University Press Lublin Poland 2008 (Polish)


[35] B Galambosi Z S Galambosi K P Svoboda and S G DeansldquoFlower yield and antioxidant properties of Arnica montana Lgrown in Finlandrdquo Drogenreport vol 11 no 19 pp 10ndash13 1998(German)

[36] B Galambosi ldquoIntroduction of Arnica montana L in FinlandrdquoZeitschrift fur Arznei- undGewurzpflanzen vol 9 no 4 pp 174ndash179 2004

[37] P Vergeer L J L van den Berg J Baar N J Ouborg and J GM Roelofs ldquoThe effect of turf cutting on plant and arbuscularmycorrhizal spore recolonisation implications for heathlandrestorationrdquoBiological Conservation vol 129 no 2 pp 226ndash2352006

[38] P Ryszka J Błaszkowski A Jurkiewicz and K Turnau ldquoArbus-cular mycorrhiza of Arnica montana under field conditionsmdashconventional and molecular studiesrdquoMycorrhiza vol 20 no 8pp 551ndash557 2010

[39] R Spitaler P D Schlorhaufer E P Ellmerer et al ldquoAltitudinalvariation of secondary metabolite profiles in flowering heads ofArnica montana cv ARBOrdquo Phytochemistry vol 67 no 4 pp409ndash417 2006

[40] U Bomme and G Daniel ldquoFirst results on selection breedingofArnica montana LrdquoGartenbauwissenschaft vol 59 no 2 pp67ndash71 1994 (German)

[41] D Sugier ldquoTrials with Arnica montana and Arnica chamissonisvar foliosa cultivation in Polandrdquo in Proceedings of the ScientificPapers of the 5th Conference on Medicinal and Aromatic Plantsof Southeast European Countries (CMAPSEEC rsquo08) pp 21ndash27Brno Czech Republic September 2008

[42] L Lovett-Doust ldquoPopulation dynamics and local specializationin a clonal perennial (Ranunculus repens) I the dynamics oframets in contrasting habitatsrdquo Journal of Ecology vol 69 no 3pp 743ndash755 1987

[43] J M Bernard ldquoLife history and vegetative reproduction inCarexrdquoCanadian Journal of Botany vol 68 no 7 pp 1441ndash14481990

[44] K Falinska ldquoGenet disintegration in Filipendula ulmaria con-sequences for population dynamics and vegetation successionrdquoJournal of Ecology vol 83 no 1 pp 9ndash21 1995

[45] B Czarnecka ldquoSpatiotemporal patterns of genets and ramets ina population of clonal perennial Senecio rivularis plant featuresand habitat effectsrdquo Annales Botanici Fennici vol 45 no 1 pp19ndash32 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


in A montana flowers and the composition of essential oilsin rhizomes and roots have been identified and characterized[19 20]

A montana is a rare plant under strict protection andis included in the IUCN Red List of Threatened Species [8]and in the Red Data Books and Red Data Lists of manyEuropean countries [21 22] Despite the loss of habitats Amontana is mainly harvested from the wild Dried flowerstraded annually in Europe are estimated to be around 50 t[23]The collection ofAmontana formedicinal purposes hasalso caused disappearance or reduction in the size of severalEuropean populations [24] The pressure on natural sourcesof this plant is alleviated by a suitable use of arnica supplyin the European region where flower heads are harvested[25] A montana is protected by law however this form ofprotection is insufficient Therefore over the last decadesother forms of active protection have appeared such asrestoration andmanagement of its natural habitats [9 26 27]plant introduction [27 28] and attempts at field cultivation[20 29 30] Biotechnological approaches for cultivation andenhancement of secondary metabolites in these species arevery interesting [31 32]

The objective of this study was to describe the impactof different ways of plant propagation and introduction onthe growth and reproduction mode of this species in thearea of its geographical range in the eastern part of Polandand to obtain information on their suitability for climaticand edaphic conditions During the six consecutive years ofthe experiment the vegetative and reproductive traits weremonitored as potentially important factors influencing theplant establishment success [33] In the future the knowledgeof the part of the life history of these species can be helpfulin cultivation which may reduce the pressure on naturalpopulation and reintroduction of A montana into theirnatural stands where the abundance of arnica has declined

2 Materials and Methods

21 Experimental Site Conditions In the present study weused individuals taken from Botanical Garden UMCS inLublin The 6-year-long experiments (2007ndash2012) were per-formed on experimental fields at the University of LifeSciences in Lublin located in the eastern part of Poland51∘331015840N 22∘441015840 E on grey-brown podsolic soil with thegranulometric composition of heavy loamy sand This sitewas chosen because the soil has physicochemical propertiescomparable to natural soil conditions for arnica [7 9]

The climate of the Lubelszczyzna region ismainly affectedby penetrating polar-oceanic masses of air and polar massesof continental air The growing season in the middle andeastern part of these region lasts 211ndash214 days [34] Theaverage month precipitation ranged from 10mm in Novem-ber 2011 to 189mm in July 2011 and the average monthtemperature ranged from minus81∘C in January 2010 to 216∘C inthe cultivation years 2007ndash2012 (Figure 1) However themeanannual sum of precipitation in the study period ranged from5102mm in 2012 to 7788mm in 2010 and the mean annualtemperature ranged from 798∘C in 2010 to 959∘C in 2008

In order to determine the edaphic conditions beforeplantation establishment topsoil samples were randomlyselected from the depth of 15 cm The main soil proper-ties were analysed The topsoil was characterized by anaverage content of organic matter (155) high phospho-rus (841mg Psdotkgminus1 PN-R-04023 1996) average potassium(722mg Ksdotkgminus1 PN-R-04022 1996+Az12002) very lowmagnesium (24mg Mgsdotkgminus1 PN-R-04020 1994+Az12004)and acid reaction (pH inH

2Omdash418 and in 1molsdotdmminus3 KClmdash

379 PN-ISO 10390 1997)

22 PlantMaterial and Experimental Design Thefield exper-iment was established in April 2007 in three ways of plantestablishment The plants were introduced as clone seedlings(CS) and rosettes (RS) and by sowing (SS)

The randomly chosen ramets taken from edge of 6-year-old genets after their natural disintegration just beforeplanting were transplanted individually into the field Theplants are marked as CS

The nine-month-old rosettes that were established afterspontaneous seed germination and originated from the col-lection subsequently they were individually transplantedinto small pits The plants are marked as RS In anotherfield experiment conducted in recent years to assess themortality rates of plants spontaneous seedling recruitmentwas observed hence the idea of using thereof for introductioninto cultivation

A montana seeds (achenes) for sowing were collectedfrom 15 individuals from the arnica collection at the endof June 2006 and stored dry and at room temperature Atthe end of April 2007 only dark and firm achenes wereselected and sown into rows where soil taken from motherplantation was scattered Our earlier experiments indicatedthat soil taken from plants compared to commonly usedstandard substrates exerted a significant impact on seedlingemergence and plant growth After sowing the soil wasslightly pressed to improve the soil contact with the seedsTen weeks after sowing when the seedlings formed rosettescharacterized by 3-4 leafs individuals were left in rows at20 cm intervals The plants are marked as SS

The A montana seeds were sown and the seedlings weretransplanted at a 40 times 20 cm distance thus ensuring almostthe same plant densities (125 plantsmminus2) The experimentalunit was made up of 75 plants The experimental design wasa randomized complete block with four replications (plotsof 6mminus2) During the vegetation period the plantation wasthree times weeded by hand We observed vegetative andreproductive traits and survival of genets from the pointof view of species biology therefore the flower heads werenot harvested Simultaneously the same characteristics werestudied in the other field experiment after harvesting flowerheads The comparable genets from the experiments withand without head harvest did not differ statistically hencethe harvest of flower heads did not determine the traitsstudied

We present changes in the characteristics studied cal-culated per genet containing several ramets formed duringthe vegetative growth In the last year of observation we


Time (years)2007 2008 2009 2010 2011 2012

020406080

100120140160180200

Mon

thly

pre

cipi

tatio

n su

m (m

m)


0510152025

minus5

minus10

Aver

age m

onth

ly te

mpe

ratu

res (

∘

C)

















3 Results



CSRSSS

0

15

5

20

40

3025

35

10

2007 2008 2009 2010 20122011

a

a

c

b

b

a

a

a

bb

bc

bb

a

b ba

Time (years)


05

1510

20

40

3025

35

cb

a

c

ba

ba

cb

a

acb

a

CSRSSS

2007 2008 2009 2010 20122011Time (years)









0

30

10

40

80

6050

70

20

a ab bb b a

b b

a abb c c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

40

20

60

80

100

120

a

bc

a bba

bc

c cb b

aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)


01

32

4

7

56 ab

b

a ba

bab

a

cb

a

a aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)





0

45004000350030002500200015001000

500

b

a

ba

ab

a

cb

a

b

cb

c

c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


002

0604

08

14

1012

ba

b aa

b

a

cb

ab

c

c

a

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)







4 Discussion



050

150100

200

350300250

abc

a

ac b

b c abb

a

bc

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

20

60

40

80

100 aa ba

ab

a

c

b

a

aaa ab cba

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)
























5 Conclusion







References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Time (years)2007 2008 2009 2010 2011 2012

020406080

100120140160180200

Mon

thly

pre

cipi

tatio

n su

m (m

m)


0510152025

minus5

minus10

Aver

age m

onth

ly te

mpe

ratu

res (

∘

C)

















3 Results



CSRSSS

0

15

5

20

40

3025

35

10

2007 2008 2009 2010 20122011

a

a

c

b

b

a

a

a

bb

bc

bb

a

b ba

Time (years)


05

1510

20

40

3025

35

cb

a

c

ba

ba

cb

a

acb

a

CSRSSS

2007 2008 2009 2010 20122011Time (years)









0

30

10

40

80

6050

70

20

a ab bb b a

b b

a abb c c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

40

20

60

80

100

120

a

bc

a bba

bc

c cb b

aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)


01

32

4

7

56 ab

b

a ba

bab

a

cb

a

a aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)





0

45004000350030002500200015001000

500

b

a

ba

ab

a

cb

a

b

cb

c

c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


002

0604

08

14

1012

ba

b aa

b

a

cb

ab

c

c

a

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)







4 Discussion



050

150100

200

350300250

abc

a

ac b

b c abb

a

bc

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

20

60

40

80

100 aa ba

ab

a

c

b

a

aaa ab cba

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)
























5 Conclusion







References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


CSRSSS

0

15

5

20

40

3025

35

10

2007 2008 2009 2010 20122011

a

a

c

b

b

a

a

a

bb

bc

bb

a

b ba

Time (years)


05

1510

20

40

3025

35

cb

a

c

ba

ba

cb

a

acb

a

CSRSSS

2007 2008 2009 2010 20122011Time (years)









0

30

10

40

80

6050

70

20

a ab bb b a

b b

a abb c c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

40

20

60

80

100

120

a

bc

a bba

bc

c cb b

aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)


01

32

4

7

56 ab

b

a ba

bab

a

cb

a

a aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)





0

45004000350030002500200015001000

500

b

a

ba

ab

a

cb

a

b

cb

c

c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


002

0604

08

14

1012

ba

b aa

b

a

cb

ab

c

c

a

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)







4 Discussion



050

150100

200

350300250

abc

a

ac b

b c abb

a

bc

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

20

60

40

80

100 aa ba

ab

a

c

b

a

aaa ab cba

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)
























5 Conclusion







References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

30

10

40

80

6050

70

20

a ab bb b a

b b

a abb c c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

40

20

60

80

100

120

a

bc

a bba

bc

c cb b

aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)


01

32

4

7

56 ab

b

a ba

bab

a

cb

a

a aa

CSRSSS

2007 2008 2009 2010 20122011Time (years)





0

45004000350030002500200015001000

500

b

a

ba

ab

a

cb

a

b

cb

c

c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


002

0604

08

14

1012

ba

b aa

b

a

cb

ab

c

c

a

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)







4 Discussion



050

150100

200

350300250

abc

a

ac b

b c abb

a

bc

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

20

60

40

80

100 aa ba

ab

a

c

b

a

aaa ab cba

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)
























5 Conclusion







References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

45004000350030002500200015001000

500

b

a

ba

ab

a

cb

a

b

cb

c

c

CSRSSS

2007 2008 2009 2010 20122011Time (years)


002

0604

08

14

1012

ba

b aa

b

a

cb

ab

c

c

a

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)







4 Discussion



050

150100

200

350300250

abc

a

ac b

b c abb

a

bc

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

20

60

40

80

100 aa ba

ab

a

c

b

a

aaa ab cba

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)
























5 Conclusion







References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


050

150100

200

350300250

abc

a

ac b

b c abb

a

bc

CSRSSS

2007 2008 2009 2010 20122011Time (years)


0

20

60

40

80

100 aa ba

ab

a

c

b

a

aaa ab cba

b

CSRSSS

2007 2008 2009 2010 20122011Time (years)
























5 Conclusion







References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















5 Conclusion







References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusion







References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Propagation and Introduction of Arnica ...downloads.hindawi.com/journals/tswj/2013/414363.pdfparasite found in mountain arnica ower heads being a limiting factor for