Effects of aqueous smoke and nitrate treatments on germination of 12 eastern Mediterranean Basin plants

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Ann. Bot. Fennici 52: 93–100 ISSN 0003-3847 (print) ISSN 1797-2442 (online) Helsinki 19 February 2015 © Finnish Zoological and Botanical Publishing Board 2015

Effects of aqueous smoke and nitrate treatments on

germination of 12 eastern Mediterranean Basin plants

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Determining the relative role of various cues on seed germination in Mediterranean plants is important to understanding their response to variable conditions. We studied germination responses of 12 eastern Mediterranean Basin species (Alyssum caricum,

A. minus, Carthamus dentatus, Daucus broteri, D. carota, Hypericum aviculariifo-lium, Muscari comosum, Onopordum caricum, Rumex crispus, Sarcopoterium spino-sum, Silene vulgaris, Smyrnium rotundifolium) to different smoke and nitrate

concen-WUDWLRQVLQODERUDWRU\6PRNHWUHDWPHQWVUHVXOWHGLQVLJQLÀFDQWLQFUHDVHLQJHUPLQDWLRQ of D. carota, O. caricum, and S. spinosum, but had a negative effect on germination of Hypericum aviculariifolium 2I WKH VWXGLHG VSHFLHV ÀYH VKRZHG VLJQLÀFDQW LPSURYHPHQWLQJHUPLQDWLRQDIWHUDWOHDVWRQHRIWKHQLWUDWHWUHDWPHQWVFRPSDUHGZLWK WKHFRQWURO,QWRWDOVPRNHDQGQLWUDWHWUHDWPHQWVVLJQLÀFDQWO\LQFUHDVHGWKHJHUPLQD-tion percentage in six of the 12 studied species. All the species that have smoke- and/or QLWUDWHVWLPXODWHGJHUPLQDWLRQZHUHSURSDJXOHSHUVLVWHUV3DEOHWRUHFUXLWDIWHUÀUH The results reveal that both smoke and nitrate improve germination of Mediterranean VSHFLHV2XUUHVXOWVDOVRVXJJHVWWKHSUHVHQFHRIVSHFLHVVSHFLÀFJHUPLQDWLRQUHVSRQVH to smoke and nitrate in Mediterranean plants.

Introduction

)LUHLVDFRPPRQGLVWXUEDQFHWKDWKDVDQVLJQLÀ-cant impact on the structure and function of plant FRPPXQLWLHV 9HUG~ 3DXVDV .HHOH\

et al. 0DQ\ SODQW VSHFLHV KDYH HYROYHG WUDLWV RU PHFKDQLVPV DOORZLQJ WKHLU SHUVLVWHQFH DIWHUÀUH([DPSOHVRIVXFKPHFKDQLVPVLQFOXGH UHVSURXWLQJ VHURWLQ\ DQG ÀUHVWLPXODWHG JHUPL-QDWLRQ.HHOH\et al.7KHODWWHULVWKHUHVXOW

of dormancy-breaking effects of heat (Thanos et

al. 1992, Herranz et al.0RUHLUD 3DXVDV DQGFKHPLFDOFRPSRXQGVIRXQGLQFKDUUHG ZRRG.HHOH\et al..HHOH\ %RQG DQGVPRNH'H/DQJH %RXFKHU'L[RQet

al. 1995, Moreira et al.,QDGGLWLRQWRLWV important role in stimulating seed germination, it has been demonstrated that chemical com-SRXQGV LQ VPRNH DOVR HQKDQFH VHHGOLQJ JURZWK (Van Staden et al. 0RUHLUD et al. 

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ÁRZHULQJ.HHOH\VRPDWLFHPEU\RJHQHVLV (Senaratna et al. 1999) and pollen germination as ZHOO DV WXEH HORQJDWLRQ 3DSHQIXV et al.  Consequently, stimulation of germination and VHHGOLQJJURZWKE\ÀUHSURGXFHGVPRNHSOD\VD key role in plant recruitment (Tormo et al.

Soils of Mediterranean-type ecosystems are XVXDOO\ FKDUDFWHUL]HG E\ WKHLU ORZ QXWULHQW FRQ-WHQW5XQGHO&RZOLQJet al.:LVKHX

et al. ,Q WKHVH HFRV\VWHPV FRQFHQWUD-tions of nitrogenous compounds in the soil (e.g., DPPRQLXPDQGQLWUDWHLQFUHDVHDIWHUÀUHV$UL-anoutsou-Faraggitaki & Margaris 1982, Stock & /HZLV&KULVWHQVHQ7KHLQFUHDVHRI these compounds in nutrient-poor Mediterranean habitats may play an important role in regulation RISRVWÀUHJHUPLQDWLRQ7KDQRV 5XQGHO Bell et al. 1999, Henig-Sever et al.

(IIHFWV RI VPRNH RQ VHHG JHUPLQDWLRQ RI Mediterranean Basin plants have received more attention in recent years (Crosti et al.  5H\HV 7UDEDXG 0RUHLUD et al.  Çatav et al.7RUPRet al.+RZHYHU there is limited information about the effect of QLWUDWH RQ VHHG JHUPLQDWLRQ ZLWK WKH H[FHSWLRQ RI IHZ VWXGLHV FRQGXFWHG LQ WKH 0HGLWHUUDQHDQ %DVLQ 'RXVVL 7KDQRV +HQLJ6HYHU et

al. 3pUH])HUQiQGH] 5RGUtJXH](FK-HYHUUtD3pUH])HUQiQGH]et al./XQD 0RUHQR 3RVLWLYH QHJDWLYH DQG QXOO effects of nitrates on germination of Mediter-UDQHDQ SODQWV ZHUH IRXQG LQ WKHVH VWXGLHV 7KLV VXJJHVWVDVSHFLHVVSHFLÀFJHUPLQDWLRQUHVSRQVH WR QLWUDWHV +RZHYHU WKH UROH RI QLWUDWHV LQ WKH germination ecology of Mediterranean plants is poorly understood due to the contrasting results obtained. Therefore, more studies are still needed to better understand the role of nitrates in germination of plant species in the Mediter-ranean Basin.

,Q WKLV VWXG\ ZH H[DPLQHG WKH JHUPLQDWLRQ responses of 12 eastern Mediterranean Basin species to different smoke and nitrate concentra-tions in laboratory condiconcentra-tions. We hypothesized that smoke and nitrate improve germination of the species studied. To test this hypothesis, VHHGV RI HDFK VSHFLHV ZHUH VXEMHFWHG WR GLIIHU-ent concGLIIHU-entrations of aqueous smoke solution, SRWDVVLXPQLWUDWH.12) and ammonium nitrate 1+12). Germination percentages and mean

JHUPLQDWLRQWLPHVRIHDFKVSHFLHVZHUHDVVHVVHG DQGWKHUHVXOWVZHUHFRPSDUHGZLWKWKRVHIRUWKH control conditions. By carrying out this experi-PHQWZHDLPHGWRWDNHDVWHSWRZDUGVÀOOLQJWKH JDSVLQRXUNQRZOHGJHRQWKHUROHRIQLWUDWHVDQG smoke as germination stimulants.

Material and methods

Study species, study area and seed collection

6HHGV RI SODQW VSHFLHV ZHUH FROOHFWHG IURP ÀUHSURQHDUHDVRI0XùOD3URYLQFHVRXWKZHVWHUQ Turkey, eastern Mediterranean Basin (Table 1). The study region has a Mediterranean climate ZLWK PLOG ZHW ZLQWHUV DQG KRW GU\ VXPPHUV 6HHGVZHUHFROOHFWHGEHWZHHQ-XO\DQG6HSWHPEHU FRLQFLGLQJZLWKWKHGLVSHUVDOSHULRGRIHDFK VSHFLHV 6HHGV ZHUH VHSDUDWHG IURP IUXLW SDUWV by hand and stored in paper envelopes at room temperature until the start of the experiment in -DQXDU\ 0HDQ VHHG PDVV ZDV GHWHUPLQHG E\ ZHLJKLQJ IRXU VDPSOHV RI VHHGV RI HDFK species.

Preparation of smoke solutions

Dry needles of Pinus brutia, a common tree spe-FLHV LQ WKH UHJLRQ ZHUH XVHG LQ SUHSDUDWLRQ RI smoke solutions. Four 5-g samples of this plant PDWHULDOZHUHKHDWHGVHSDUDWHO\LQJODVVÁDVNVLQ WKHRYHQIRUPLQDW&7KHPRXWKRIHDFK ÁDVNZDVWLJKWO\FRYHUHGZLWKDQDOXPLQLXPIRLO to capture the smoke generated from the heated QHHGOHV 6XEVHTXHQWO\ PO RI GLVWLOOHG ZDWHU ZDVSRXUHGRYHUWKHKHDWHGPDWHULDODQGOHIWIRU PLQXWHVdDWDYet al. %\WKLVSURFHGXUH the active chemicals found in smoke are dissolved LQ ZDWHU -lJHU et al. 7KH VROXWLRQV ZHUH ÀOWUDWHG IURP WKH ÁDVNV LQWR D ERWWOH WR REWDLQ D FRQFHQWUDWHGVPRNHVROXWLRQS+

The experiment

The germination-medium compositions are JLYHQ LQ 7DEOH 7ZHQW\ÀYH VHHGV RI HDFK

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VSHFLHV ZHUH VRZQ GLUHFWO\ LQ 3HWUL GLVKHV FRQ-WDLQLQJ PO RI WKH PHGLD (DFK WUHDWPHQW FRQVLVWHG RI IRXU UHSOLFDWHV 3HWUL GLVKHV ZHUH WKHQSODFHGLQDQLQFXEDWRUDW&LQGDUN-QHVV 3UHYLRXV VWXGLHV VKRZHG WKDW WKHVH FRQGL-tions are favourable for germination of many Mediterranean-climate species (Bellairs & Bell /XQDet al.7KHVHHGVZHUHFKHFNHG for germination under a stereomicroscope every GD\VGXULQJWKHÀUVWZHHNVDQGWKHQZHHNO\ until the end of the experiment. It should be QRWHG WKDW WKH VHHGV ZHUH H[SRVHG WR GD\OLJKW IRU VKRUW GXUDWLRQV ² PLQXWHV GXULQJ WKH germination checks. The criterion of germination ZDVYLVLEOHUDGLFOHSURWUXVLRQ*HUPLQDWHGVHHGV ZHUHFRXQWHGDQGUHPRYHGIURPWKH3HWULGLVKHV

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Data analysis

After removing empty seeds, the remaining seeds ZHUH WKHQ VFRUHG DV JHUPLQDWHG RU QRQJHUPL-QDWHG )RU HDFK VSHFLHV WKH ÀQDO SURSRUWLRQ RI JHUPLQDWHG VHHGV LQ HDFK WUHDWPHQW ZDV FRP- SDUHGZLWKWKHFRQWUROXVLQJWKHDQDO\VLVRIGHYL-DQFHZLWKDELQRPLDOHUURUGLVWULEXWLRQ0RUHLUD

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D)/kZKHUHn is the number of seeds that germi-nated on day D, D is the number of days from the beginning of the incubation period, and k is the total number of seeds that germinated during the incubation period. For each species, the dif-IHUHQFHV EHWZHHQ WKH PHDQ JHUPLQDWLRQ WLPH LQ WKH FRQWURO DQG WUHDWPHQW JURXSV ZHUH DQDO\]HG XVLQJ RQHZD\ $129$ IROORZHG E\ 'XQQHWW·V test for multiple comparisons. Homogeneity of YDULDQFH ZDV YHULÀHG ZLWK %DUWOHWW·V WHVW EHIRUH each analysis. Because of the large number of SDLUZLVH FRPSDULVRQV WKH VLJQLÀFDQFH OHYHO IRU DOODQDO\VHVZDVVHWDWp This approach is less conservative than the Bonferroni correction 0RUDQ DQG KDV EHHQ HIIHFWLYHO\ XVHG LQ many studies in recent years (e.g. Moreira et al. 7DYüDQRùOX *UNDQ

Results

The germination percentages in the controls varied among the study species, ranging from

WR7DEOH,QIRXUVSHFLHVCartha-mus dentatus, Muscari comosum, Onopordum caricum, Smyrnium rotundifolium) the dormancy

OHYHO ZDV YHU\ KLJK JHUPLQDWLRQ LQ WKH

FRQWUROZKHUHDVDQRWKHUIRXUVSHFLHVAlyssum

caricum, A. minus, Rumex crispus, Silene vul-garisKDGYHU\ORZOHYHOVRIGRUPDQF\! germination in the control). Smoke and nitrate treatments failed to break high-level dormancy (zero germination in the control) in Muscari

comosum and Smyrnium rotundifolium.

Moreo- YHUJHUPLQDWLRQLQWKHWKUHHVSHFLHVZLWKQRGRU-mancy (Alyssum minus, Rumex crispus, Silene

vulgaris ZDV QRW DIIHFWHG E\ DQ\ RI WKH WUHDW-PHQWV7DEOH

6PRNH WUHDWPHQWV UHVXOWHG LQ D VLJQLÀFDQW increase in germination of three species, but affected negatively germination of Hypericum

aviculariifolium 7DEOH 2I WKH VSHFLHV VWXGLHGÀYHVKRZHGVLJQLÀFDQWLPSURYHPHQWLQ germination after at least one of the nitrate treat-PHQWV DV FRPSDUHG ZLWK WKH FRQWURO 7DEOH Regarding the effect of nitrates on germination, P0.12ZDVWKHPRVWHIIHFWLYHWUHDWPHQW LQZKLFKJHUPLQDWLRQRIÀYHVSHFLHVZDVVWLPX-ODWHG7KHUHPDLQLQJQLWUDWHWUHDWPHQWVZHUHWKH OHDVWHIIHFWLYHRQHVDVJHUPLQDWLRQRIRQO\WZR VSHFLHV ZDV VWLPXODWHG LQ HDFK +RZHYHU QRQH of the nitrate treatments affected germination of WKHVWXG\VSHFLHVQHJDWLYHO\7DEOH

Germination response of the species to VPRNHDQGQLWUDWHWUHDWPHQWVYDULHG2QO\LQWZR species (Onopordum caricum and Sarcopoterium

spinosum JHUPLQDWLRQ ZDV LPSURYHG ERWK E\

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smoke and nitrate. The remaining species had different responses to smoke and nitrate treat-ments. For example, germination of Carthamus

dentatus and Daucus broteri ZDV HQKDQFHG E\ nitrate but not by smoke; inversely, germination percentage in Daucus carotaZDVLQFUHDVHGRQO\ E\VPRNH1LWUDWHDSSOLFDWLRQVGLGQRWGHFUHDVH WKH JHUPLQDWLRQ SHUFHQWDJH LQ FRPSDULVRQ ZLWK that in the control in Hypericum

aviculariifo-liumWKHRQO\VSHFLHVLQZKLFKJHUPLQDWLRQZDV negatively affected by smoke. An association EHWZHHQ UHJHQHUDWLRQ PHFKDQLVP RI VSHFLHV and their germination response to smoke and/ RUQLWUDWHWUHDWPHQWVZDVIRXQG7KDWLVWKHVSH-FLHV ZLWK JHUPLQDWLRQ HQKDQFHG E\ VPRNH DQG RUQLWUDWHZHUHDOOSURSDJXOHSHUVLVWHUV3DQG QRQVWLPXODWHG VSHFLHV ZHUH DOO SURSDJXOH QRQ SHUVLVWHUV3²H[FHSWRQHLQZKLFKUHJHQHUDWLRQ PHFKDQLVPLVXQNQRZQ7DEOH

Apart from Daucus broteri LQ ZKLFK 0*7 LQFUHDVHGDIWHUDVPRNHWUHDWPHQWVPRNH WUHDWPHQWV GLG QRW VLJQLÀFDQWO\ FKDQJH 0*7 LQ WKH UHPDLQLQJ VSHFLHV 7DEOH 

Sarcopote-rium spinosum JHUPLQDWHG VLJQLÀFDQWO\ IDVWHU LQWKHORZHUFRQFHQWUDWLRQVRIQLWUDWHWKDQLQWKH control. On the other hand, nitrate treatments UHVXOWHG LQ VORZHU JHUPLQDWLRQV LQ Onopordum

caricum and Daucus broteri 1R VLJQLÀFDQW FKDQJHV LQ 0*7 ZHUH IRXQG LQ RWKHU VSHFLHV after the nitrate treatments.

Discussion

7KH VPRNH DQG QLWUDWH WUHDWPHQWV VLJQLÀFDQWO\ increased the germination percentages in six of the 12 studied species (Alyssum caricum,

Carthamus dentatus, Daucus broteri, D. carota, Onopordum caricum and Sarcopoterium spino-sum). Of the study species, only germination

of Hypericum aviculariifolium ZDV QHJDWLYHO\ DIIHFWHG E\ VPRNH $V IDU DV ZH NQRZ RXU UHVXOWVSURYLGHWKHÀUVWHYLGHQFHRIVPRNHDQG nitrate-stimulated germination in Onopordum

caricumZKLFKLVHQGHPLFWR7XUNH\7KLVUHVXOW may have implications to in-situ and/or ex-situ conservation of endemic plants in Mediterranean vegetation in Turkey.

Availability of nitrogenous compounds can LQFUHDVHLQWKHVRLOGXULQJWKHÀUVWUDLQ\VHDVRQ IROORZLQJÀUH7KDQRV 5XQGHOODQGLQ 0HGLWHUUDQHDQ UHJLRQV SUROLÀF JHUPLQDWLRQ RI many plants generally occurs during this period (Thanos & Georghiou 1988, Bell et al. 1995). Consequently, many researchers focused on the UROH RI QLWURJHQRXV FRPSRXQGV LQ SRVWÀUH JHU-mination (Thanos & Rundel 1995, Bell et al. 1999, Henig-Sever et al. 7KH UHVXOWV RI those studies indicate that nitrate and ammo-nium can play an important role in germination of several plants (e.g., Emmenanthe

penduli-ÁRUD, 3KDFHOLD JUDQGLÁRUD +RZHYHU .HHOH\

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DQG )RWKHULQJKDP UHSRUWHG WKDW .12 solutions indeed stimulate germination in E.

pen-GXOLÁRUD and 3JUDQGLÁRUDEXWRQO\DWORZS+

Accordingly, they proposed that both oxidizing gases in smoke and acids are potential germi-QDWLRQ WULJJHUV IRU WKHVH SRVWÀUH DQQXDOV 2Q WKH RWKHU KDQG 3pUH])HUQiQGH] et al.  VKRZHG WKDW JHUPLQDWLRQ RI WKHLU VWXG\ VSHFLHV PDLQO\ KHUEDFHRXV SODQWV ZDV QRW DIIHFWHG E\ the concentration of H_{ZLWK RU ZLWKRXW WKH}

nitrate ion. Although our study cannot be com- SDUHGZLWKWKHDERYHRQHVVLQFHRXUH[SHULPHQ-tal design did not include a pH gradient, our results indicate that germination success of spe-cies is variable in smoke and nitrate treatments at the same pH level (in our case pH = 5.8).

The mechanism of improving seed germina-WLRQE\QLWURJHQRXVFRPSRXQGVZDVLQYHVWLJDWHG by Henig-Sever et al. 7KH UHVXOWV RI their study suggest that nitrate increases the seed sensitivity to GA JLEEHUHOOLF DFLG ZKHUHDV ammonium directly affects lipase activity. Ger-mination induction by nitrates is achieved in the FRQFHQWUDWLRQUDQJHXSWRP0ZKLOHKLJKHU concentrations have a negative effect (Thanos & Rundel 1995, Bell et al.3pUH])HUQiQGH] 5RGUtJXH](FKHYHUUtD 7KH FRQFHQWUD-WLRQV ZH DSSOLHG LQ RXU VWXG\ ZHUH ZLWKLQ WKLV UDQJH VR WKLV FRXOG H[SODLQ ZK\ ZH IRXQG QR negative effects of any nitrate treatment on the germination of the studied species.

7KH DVVRFLDWLRQ EHWZHHQ WKH FDSDFLW\ WR UHFUXLW DIWHU ÀUH 3 3² DQG JHUPLQDWLRQ response of species suggests that smoke- and nitrate-stimulated germination are impor-tant determinants of the regeneration strategy in Mediterranean plants. In the Mediterranean %DVLQ ÀUHSURQH SODQW FRPPXQLWLHV DUH SK\-logenetically and phenotypically clustered as a UHVXOW RI WKH ÀOWHULQJ SURFHVVHV RI ÀUH SURFHHG-LQJRQWKHVSHFLHVWKDWKDYHÀUHSHUVLVWHQWWUDLWV 3DXVDV 9HUG~ ,W KDV EHHQ VKRZQ WKDW ÁDPPDELOLW\HQKDQFLQJWUDLWVLQSRVWÀUHVHHGHUV 3YDU\DGDSWLYHO\LQUHVSRQVHWRÀUH0RUHLUD

et al E\ SURGXFLQJ WKH QHFHVVDU\ FXHV for triggering germination from the seed bank and opening spaces for the regeneration of the RIIVSULQJ 3DXVDV 0RUHLUD 0RUHRYHU SURSDJXOH SHUVLVWHQFH 3 LV DQ HYROXWLRQDU\ conserved trait at the genus and family levels

since the appearance of Mediterranean climate LQWKHUHJLRQ9HUG~ 3DXVDV7KHUHIRUH PDQ\ IDPLOLHV LQ ZKLFK 3 WUDLW LV REVHUYHG LQFOXGH VSHFLHV ZLWK HQKDQFHG SRVWÀUH JHUPL-nation (Çatav et al. DQG SRVWÀUH HVWDE-OLVKPHQW 7DYüDQRùOX *UNDQ 3DXOD DQG3DXVDVDOVRGHPRQVWUDWHGWKHKLJKHU FDSDFLW\ RI ÀUH VWLPXODWHG JHUPLQDWLRQ LQ QRQ UHVSURXWHUV5²WKDQLQUHVSURXWHUV5+RZ-HYHU VLQFH RXU VWXG\ LQFOXGHG MXVW D GR]HQ species, the results on regeneration mechanism should be considered carefully.

In summary, our results reveal that both smoke and nitrate play a role in germination stimulation of Mediterranean species, at least for the subset of the studied species. Our results DOVR VXJJHVW WKH SUHVHQFH RI VSHFLHVVSHFLÀF germination response to smoke and nitrates in Mediterranean plants.

Acknowledgments

7KHVHHGVZHUHFROOHFWHGLQWKHÀHOGZLWKWKHSHUPLVVLRQRI the Ministry of Food, Agriculture and Livestock of Republic RI 7XUNH\ QR GDWH 7KH H[SHULPHQWV ZHUHFRQGXFWHGLQWKHODERUDWRULHVRI'HSDUWPHQWRI%LRORJ\ 0XùOD 8QLYHUVLW\ 7XUNH\ 7KH ÀHOGZRUN RI WKLV VWXG\ ZDV VXSSRUWHGE\+DFHWWHSH8QLYHUVLW\6FLHQWLÀF5HVHDUFK8QLW SURMHFWQR'

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