ALLELOPATHIC POTENTIAL OF SUNFLOWER ROOTS AND ROOT EXUDATES 1. Introduction

ALLELOPATHIC POTENTIAL OF SUNFLOWER ROOTS AND ROOT EXUDATES 1 D. Ciarka 1., H. Gawronska, M. Malecka, S.W. Gawronski Department of Pomology and Basic Natural Sciences in Horticulture, Faculty of Horticulture and Landscape Architecture, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warsaw, Poland 1 Corresponding author: ciarka@alpha.sggw.waw.pl Introduction Sunflower, well know from its high allelopathic activity [LEATHER 1987; NARWAL et al. 1999a; BATISH et al. 2002], produces many chemical compounds, which affect germination, growth and development of other plants [MACIAS et al. 1999]. Plant organs differ in allelopathic potential [WÓJCIK-WOJTKOWIAK et al. 1998] and the presence of allelochemicals in the above ground organs of sunflower, is well documented [LEATHER 1983a, b; MACIAS et al. 1999; NARWAL, et al. 1999a], but complex data on allelopathic potential of particular organs of sunflower and especially of roots and roots exudates are limited [IRONS and BURNSIDE 1982; LEATHER 1983a, b]. This work is continuation of studies on allelopathic potential of different organs of 37 genotypes of sunflower against mustard [CIARKA et al. 2002a], against common in Europe weeds [CIARKA et al. 2002b] and of 10 genotypes against winter wheat [CIARKA et al. 2002a]. Results from our field experiments with plots where only sunflower roots were left, showed significant reduction in weed infestation and wheat stand [GAWRONSKI 2003]. This suggests that roots of sunflower possess allelopathic activity but preliminary study with root extracts did not confirm that. Our working hypothesis was that sunflower allelochemicals were present in the soil due to either: 1/ earlier exudation to the rhizosphere by living roots, 2/ these compounds were present in dying roots that were left in the soil after plant harvest, which became active in the rhizosphere during roots decomposition, or what seems most possibly 3/ due to both. The objectives of this work therefore, were to: (i) estimate allelopathic activity of sunflower roots, (ii) check whether soil, after sunflower cultivation, contained compounds of allelopathic activity, and to (iii) study if roots of sunflower exude substances of allelopathic activity. 1

Materials and methods The allelopathic activity of sunflower roots and of roots exudates was evaluated in 3 separate experiments. In the first, effects of allelochemicals contained in water extracts from sunflower roots on germination of mustard and winter wheat used in our study as model plants for dicotyledonous weeds and for a crop - was evaluated. Ten cvs. of sunflower: Albena, Giganta, Lech, Ogrodowy, Optisol, Pardisol, Pastewny, Polizie, Printasol, Wielkopolski and a breeding line - 57958 were grown in the pots (25 cm ) filled with 10 l of substrate (peat moss and sand in proportion 1:3, supplemented with CaCO 3 and nutrients of MIS 4). Plants were watered daily in excess and agricultural practices were as recommended for sunflower. At flowering, plants were harvested and roots were carefully collected when soil was sieved through 2 mm mesh sieve. Roots were air-dried, ground into a fine powder, which was than soaked for 24 h in water, and the extract after filtration was directly used in germinating tests. Seeds of mustard (Sinapis alba L.) cv. Nakielska and of wheat (Triticum aestivum L.) cv. Zyta were germinated in Petri dishes on filter paper, moistened with 5ml of extracts in concentrations: 2.5, 5.0, 7.5 and 10.0 % DM w/v and with distilled water (control) in darkness at 20 C for 14 days, in control cabinet. Germinating seeds were counted daily and removed. For each cv. six replicates were used with 50 seeds per replication (Petri dish). Results are presented in % sown seeds and for the dynamic of germination Piper Index (PI) was also calculated [PIPER 1952 after GRZESIUK and KULKA 1981]. The effect of allelopathic compounds contained in soil, which was previously used for sunflower cultivation, was evaluated by growing, in this soil, mustard plants (experiment 2 nd ). Four cvs. of sunflower: Albena, Pardisol, Pastewny, and Wielkopolski were grown in the pots as above. At harvest, any visible roots were taken out; the soil was sift out through sieve and right away was used for mustard cultivation. Substrate never used for cultivation was a control. Mustard seeds were sown into pots of 10 cm (10 seeds pot -1 ) in 10 replicates for each sunflower cv. Plants were grown in greenhouse and harvested after 28 days of cultivation and the fresh weights of above ground organs and of roots were recorded. In the experiment 3, allelopathic activity of root exudates was checked by simultaneous incubation of mustard seeds and seedlings together with sunflower seedlings. Five seedlings 2

(four-day old) of sunflower Lech and Ogrodowy cvs. (separately) were placed in Petri dishes, layered with blotting paper and moistened with distilled water. Three days later 10 seeds of mustard were added to each Petri dish. Incubation conditions were as described in the experiment 1. Length of mustard hypocotyls and of roots were recorded on day 4. Experiment was run in 8 replications for each sunflower cultivar. All data were analysed with ANOVA and presented data are mean ± SE, n = 6, 10, 8 depending on experiment. HSD of Tukey test and LSD of t - Student test at p < 0.05 are provided where differences between combinations were significant. Results and discussion Germination of mustard was only slightly and of wheat almost not affected by aqueous extracts of sunflower roots extracts. Since for germination, the differences between used concentrations were relatively small (though in case of mustard significant for extracts of 5 cvs.), data for germination are presented as average of all concentrations (Fig. 1 and 2). Values in the bars illustrate the range of germination in dependence of concentrations with higher and lower numbers referring to the lowest and highest concentrations respectively. Out of 11 tested genotypes only extracts of Pardisol roots reduced germination, on average, by more than 10 % (88.5, 87.3 and 83.3 % of sown seeds germinated at concentrations 5.0, 7.5, and 10.0 % DM w/v, respectively). Also Printasol showed similar effect but only at the highest extract concentration (88.8 % of sown seeds germinated). Mustard germination in the presence of root extracts of all the other cvs. and at every concentration ranged between 90.5 and 97.8 % which was very similar to germination in the control - H 2 O (96.5-99.5 %). In case of wheat germination, allelopathic effect of sunflower roots was not observed at all. Even at the highest concentration between 95.7 and 100.0 % of sown seeds germinated, being almost at the same level as in the control (99.3-100.0 %), Fig. 2. These results are in a range of expectation since, as shown in our earlier study with 10 cvs. of sunflower, leaf extracts (of highest, among all sunflower organs, allelopathic activity), at the highest concentration, have a very slight, if any, allelopathic effect on winter wheat germination [CIARKA et al. 2002a]. It should be mentioned that the former experiments were performed with the same cv. of winter wheat and under the same, as in this work, laboratory conditions. A slightly higher allelopathic effect was observed in the dynamics of seed germination but 3

again this effect was more evident in case of mustard. According to Pipper Index, one mustard seed needed 1.98 to 2.42 days to germinate when cultured in H 2 O, while in the presence of root extracts, this value ranged between 2.03 and 4.23 days depending on the sunflower genotype and extract concentration (Fig. 3). For every genotype, the delay in germination showed a concentration dependent manner being greatest for highest concentration (Fig. 3, Tab. 1). Extracts of cv. Pardisol. were, at highest concentration, most effective as under this condition one seed to germinate needed 2.25 days more than in H 2 O (1.98 vs. 4.23), (Tab. 1) though, in all concentrations the most effective was the breeding line 57859. Allelopathic effects of sunflower root extracts on the dynamic of wheat germination showed different, than in the case of mustard, pattern as it was both of inhibitory and stimulatory mode of action. The inhibitory effect was recorded in the case of 8 genotypes (but significant only in 4) with PI values ranging depending on cv. from 1.72 to 2.04 d vs. 1.71 d in the control (Fig. 4, Tab. 2). This effect was very weak comparable to mustard, in which the greatest delay when compared to the control was 2.25 d while in the case of wheat only 0.33 d (Fig. 4, Tab. 2). Stimulatory effect was manifested by an acceleration of wheat germination, which took place in the case of 5 sunflower genotypes (PI of control 1.71 vs. 1.41 1.70 d) (Fig. 4, Tab. 2). The effect of root extracts on dynamic of germination showed concentration dependent manner being greater for higher concentration in inhibition while the opposite was true for the speeding -up of germination as the greater acceleration was recorded at the lowest concentration (Tab. 2). The differences between combinations (including H 2 O) were mostly significant (Tab. 1 and 2). These results clearly showed that aqueous extracts of sunflower roots, contrary to all other organs, possessed a very slight, if any, allelopathic activity. As shown in another of our studies, the above ground organs of sunflower plant possess a substantially higher allelopathic activity [CIARKA et al. 2002a]. Among the tested organs, leaves have the highest allelopathic activity, which on average for all 37 tested genotypes and 4 concentrations, reduced mustard germination to 55.0% of sown seeds. Moreover, at the highest concentration of 15 sunflower genotypes, mustard germinated below 10 %. Evidently a lower allelopathic activity, though still higher than roots, have stems and inflorescence, which reduce germination (again on average for cvs. and concentrations) to 79.85 and 81.4 % of sown seeds respectively [CIARKA et al. 2002a]. The highest allelopathic potential of leaves and higher of above ground organs than of roots seem to be common for most species [WÓJCIK-WOJTKOWIAK et al. 1998]. However, there are 4

in literature data that roots of some plant species are the source of compounds of high allelopathic activity [OLESZEK 1992; HARBORNE 1997; BERTIN et al. 2003]. Low roots activity, recorded in this work, not necessarily mean that, sunflower roots do not contain allelocompounds although, it cannot be completely ruled out. It is also possible that compounds of allelopathic activity in sunflower roots might be not soluble in water and if so, they will not be present in water extracts. In this study, only water extraction as closer to existing in nature extraction were used. Also, allelocompounds might be present in the roots as non-active and when exuded into the rhizosphere they would became active via the concerted action with microorganisms. Finally, allelochemicals could be present in the root parts that are not accessible for collection under this method of cultivation. The last two possibilities were also tested in this work, in experiments in which either mustard was grown in the soil that was previously used for sunflower cultivation and then deprived of any visible roots or in experiment with mustard seedlings co-cultured in Perti dish together with sunflower seedlings (see below). Growth of mustard plants was significantly inhibited by compounds contained in the soil, most probably both exudes of sunflower roots and/or tiny, not possible to collect roots and root hairs (Fig. 5). This inhibition was true for all four cultivars. As seen in Table 3 the total plant biomass, on average for all 4 cvs. was reduced by 83.1% as compared to control with Pastewny cv. being the most effective (85.1 %). Similarly an inhibition of growth of soybean, sorghum and sunflower [IRONS and BURNSIDE 1982] and of sorghum, pearl millet, maize and cotton [NARWAL et al. 1999b] when these plants were grown in soil previously used for sunflower cultivation was observed. However, NARWAL et al. [1999b], in the 2 nd year of study, instead of inhibition a stimulation of growth have observed. The inhibitory effect was more evident in case of above ground organs, for which the reduction ranged from 90.8 % to 93.7 % while for roots it ranged between 40.7% and 61.5 % (Fig. 5, Tab. 3). Different reduction of biomass accumulated by shoots and by roots lead to substantial change in the root:shoot ratio, which in the control equals 0.31, while, in plants grown in the soil after sunflower cultivation it ranged between 1.31 and 2.91 (Tab. 3). CASPERSEN et al. [1999] also recorded a lower biomass accumulation by lettuce plants treated with ferulic acid, a phenolic compound showing allelopathic activity [POLITYCKA 1996] and is present in sunflower [BATISH et al. 2002]. However contrary to us, roots were more than shoot affected which might be due to higher relative humidity under which lettuce plants were grown (culture flask, climate chamber vs. greenhouse) and this could lead to lower transport 5

of phenolic compounds to the shoot and consequently to smaller inhibitory effect on a shoot CASPERSEN et al. [1999]. Results from this experiment provided information that soil, after sunflower cultivation, contained compounds of allelopathic activity but did not answer on a question whether sunflower roots exude allelopathics. To answer on this question mustard seedlings were cultured in the presence of, more advanced in development, seedlings of sunflower. Figure 6 shows that mustard seedlings were less vigorous when grown together with sunflower, length of which was reduced by 46.3 and 64.2 %. Allelopathic - inhibitory effect was more pronounced on hypocotyls than on roots (51.1 and 78.2 % vs. 40.8 and 47.9 %) which very well corresponded with results described earlier (Fig. 6vs. Fig.5 and Tab. 3), in which a shoot was also more affected than roots by allelocompounds present in the soil. In reduction of both organs cv. Ogrodowy was more effective than Lech (Fig. 6). This clearly demonstrated that sunflower seedlings affected mustard seedlings growth via substances exuded by roots although the volatilisation of some compounds either by roots or epicotyls or both cannot be excluded. These data are in agreement with results of IRONS, BURNSIDE [1982] and of LEATHER [1983a, b] who also observed allelopathic activity of sunflower root exudates. According to BERTIN et al.[2003] and references therein] roots exudates play, among others, an important role as allelochemicals in the rhizosphere as it was shown for black walnut, wheat, and sorghum. It also refers to buckwheat, known for a high allelopathic potential [GOLISZ et al. 2002], which also reduced growth of mustard seedling but the effect was much slighter and moreover, contrary to this work, roots were more strongly inhibited than hypocotyls [GOLISZ, personal communication]. This suggests that allelocompounds of sunflower differ in the mode of action from those of buckwheat, most possibly due to different chemical structure and nature. Conclusions: 1. Compounds contained in aqueous extracts of sunflower roots, accessible for collection, have a very low if any, allelopathic activity on mustard and wheat germination. 2. Allelochemicals derived from sunflower roots differ in the mode of action on dynamics of germination of mustard and wheat being inhibitory in mustard but both inhibitory and stimulatory in the case of wheat. 3. Soil after sunflower cultivation, deprived of any visible sunflower roots, contained 6

compounds of all allelopathic activity most probably roots exudates and not possible to be collect root parts. 4. Sunflower seedlings exude compounds of allelopathic inhibitory - activity against mustard seedlings. References BATISH D. R., TUNG P., SINGH H. P., KOHLI R. K. 2002. Phytotoxicity of sunflower residues against some summer season crops. J. Agronomy & Crop Sci. 188: 19-24. BERTIN C., YANG X., WESTOM L. A. 2003. The role of root exudates and allelochemicals in the rhizosphere. Plant and Soil 256: 67-83. CASPERSEN S., SUNDIN P., MUNRO M., ADALSTEINSSON S., HOOKER J. E., JENSEN P. 1999. Interactive effects of lettuce (Lactuca sativa L.), irradiance, and ferulic acid in axenic, hydroponic culture. Plant and Soil 210: 115-126. CIARKA D., GAWRONSKA H., PRAWICZ U., GAWRONSKI S.W. 2002a. Genotypical differences in allelopathic potential of sunflower. Abstract of Third World Congress on Allelopathy: Challenge for the New Millennium, Tsukuba, Japan, 26-30 VIII 2002.: 78. CIARKA D., GAWRONSKA H., GAWRONSKI S. W. 2002b. Weed species reaction to sunflower allelopathics. Abstract of Third World Congress on Allelopathy: Challenge for the New Millennium Tsukuba, Japan, 26-30 VIII 2002: 162. GAWRONSKI S.W. 2003. Allelopathy as a strategy for weed control in organic farming. Abstract of Fifth International Conference, Ecophysiological Aspects of Plant Responses to Stress Factor Cracow, Poland. Acta Physiol. Plant. 25, (3), p.:25. GOLISZ A., CIARKA D., GAWRONSKI S.W. 2002. Allelopahtic activity of buckwheat Fagopyrum esculentum Moench. Abstract of Third World Congress on Allelopathy: Challenge for the New Millennium, Tsukuba, Japan, 26-30 VIII 2002: 78. HARBORNE J. B. 1997. Ekologia biochemiczna. Tlum. W. Oleszek. PWN, Warszawa351 ss. 7

IRONS S.M., BURNSIDE O.C. 1982. Competitive and allelopathic effects of sunflower (Helianthus annuus). Weed Sci. 30: 372-377. LEATHER G.R. 1983a. Weed control using allelopathic crop plants. J. Chem. Ecol. 9, 8: 983-989. LEATHER G.R. 1983b. Sunflower (Helianthus annuus) are allelopathic to weeds. Weed Sci. 31: 37-42. LEATHER G.R. 1987. Weed control using allelopathic sunflower and herbicide. Plant and Soil 98:17-23. MACIAS F.A, GALINDO C.G., MOLINILLO J.M., CUTLER H.G. 1999. Bioactive compounds from the genus Helianthus. In: Recent advances in allelopathy. Volume I: A Science for the Future. F.A.Macias, J.C.G.Galindo., J.M.G.Molinillo, H.G. (eds.). Servicio De Publicaciones-Universidad De Cadiz, Puerto Real, Cadiz, Spain.: 121-148. NARWAL S.S., SINGH T.,. HOODA J.S, KATHURIA M.K. 1999a. Allelopathic effects of sunflower on succeeding summer crops. 1. Field studies and bioassays. Allelopathy J. 6(1): 35-48. NARWAL S.S.,.SINGH T, HOODA J.S., KATHURIA M.K. 1999b. Allelopathic effects of sunflower on succeeding summer crops.2. Pot culture and biomass decomposition. Allelopathy J. 6(2): 209-226. OLESZEK W. 1992. Techiniki badan allelopatii. Wiadomosci Botaniczne 36, (3/4): 17-25. PIPER. H. Das Saatgut. Berlin, cited after: Grzesiuk S. and K. Kulka1981. Fizjologia i biochemia nasion. PWRiL: 374-376. POLITYCKA B. 1996. Peroxidase activity and lipid peroxidation in roots of cucumber seedlings influenced by derivatives of cinnamic and benzoic acids. Acta Physiol. Plant. 18(4): 365-370. 8

WÓJCIK WOJTKOWIAK D., POLITYCKA B., WEYMAN KARCZMARKOWA W. 1998. Allelopatia. Wydawnictwo AR w Poznaniu:99 ss. Acknowledgements: This research were financially supported by: 1/ European Commission, 5FP # QLK5-CT-2000-01418 2/ State Committee for Scientific Research, KBN # 117/E-385/SPUB-M/5PRUE Keys words: Allelopathy, root aqueous extracts and exudates, germination, growth seedling, sunflower, mustard, wheat. Summary In this work we studied the allelopathic effects of aqueous roots extracts of 11 cvs. of sunflower on mustard and wheat germination. We also checked whether soil, after sunflower cultivation, contained compounds of allelopathic activity against mustard plants, and if, roots of sunflower seedlings exude substances of allelopathic activity on co-cultured mustard seedlings. Compounds contained in aqueous extract of roots possessed low (mustard) if any (wheat) allelopathic inhibitory effect on seed germination. Allelochemicals derived from sunflower roots differ in the mode of action on dynamics of germination being inhibitory in mustard but both inhibitory and stimulatory in the case of wheat. Contrary, mustard plant growth was strongly inhibited by compounds that were in the soil, previously used for sunflower cultivation, most probably root exudates and not possible to collect root parts. Growth of mustard seedlings co-cultured sunflower seedlings was also inhibited most possibly due to exudation, by sunflower roots, substances of allelopathic activity. Inhibitory effects of sunflower on mustard growth were more pronounced on mustard hypocotyls and shoots than on roots. 9

ALLELOPATYCZNY POTENCJAL KORZENI SLONECZNIKA ZWYCZAJNEGO (Helianthus annuus L.) Ciarka D., Gawronska H., S.W. Gawronski Katedra Sadownictwa i Przyrodniczych Podstaw Ogrodnictwa, Wydzial Ogrodnictwa i Architektury Krajobrazu, SGGW, Nowoursynowska 166, 02-787 Warszawa, Polska Slowa kluczowe: Allelopatia, wodne wyciagi z korzeni, wydzieliny korzeniowe, kielkowanie wzrost roslin, slonecznik, gorczyca, pszenica. Streszczenie W pracy (i) oceniono wplyw wodnych wyciagów korzeni 11 odmian slonecznika na kielkowanie gorczycy i pszenicy ozimej, (ii) sprawdzono czy gleba, w której uprzednio uprawiano slonecznik, zawiera allelozwiazki oraz (iii) zbadano, czy wydzieliny korzeniowe siewek slonecznika oddzialywaja allelopatycznie na siewki gorczycy bialej. Badania wykazaly, ze wodne wyciagi korzeni slonecznika, wszystkich badanych odmian, w niewielkim stopniu hamowaly (gorczyca) lub prawie w ogóle (pszenica) nie hamowaly kielkowania nasion. Bardziej widoczny byl wplyw na czas kielkowania z tym, ze w przypadku gorczycy czas kielkowania zawsze sie wydluzal, podczas gdy u pszenicy notowano zarówno opóznienie jak i przyspieszenie kielkowania. Bardzo wyrazny (statystycznie udowodniony) efekt allelopatycznego oddzialywania na wzrost i akumulacje biomasy u gorczycy stwierdzono, gdy rosliny rosly w glebie, w której uprzednio uprawiano slonecznik (po wczesniejszym usunieciu wszystkich widocznych korzeni slonecznika). Wskazuje to, iz gleba po uprawie slonecznika zawierala albo wydzieliny korzeniowe o wlasciwosciach allelopatycznych lub zwiazki te znajdowaly sie w drobnych (niemozliwych do pozyskania z uprawy w glebie) korzeniach i wlosnikach, a uwalnianych do gleby podczas rozkladu korzeni. Istotne hamowanie wzrostu gorczycy notowano takze, gdy siewki gorczycy hodowano razem z siewkami slonecznika wydzielajacymi do podloza substancje allelopatyczne, przy czym hypokotyl, podobnie jak czesc nadziemna w poprzednim doswiadczeniu, byl silniej hamowany niz korzen. 10

Fig. 1. Germination of white mustard (Sinapis alba L.) cv. Nakielska in the presence of allelochemicals contained in aqueous root extracts of 11 cultivars of sunflower (Helianthus annuus L.). Bars marked by different letters differ significantly according to HSD Tukey test at p < 0.05. indicate significant difference from control as estimated by the t- Student s test at p < 0.05. Values in bars illustrate range in germination for all used concentrations. Rys. 1. Kielkowanie gorczycy bialej (Sinapis alba L.) odm. Nakielska w obecnosci wodnych wyciagów korzeni 11 odmian slonecznika zwyczajnego (Helianthus annuus L.). Rózne litery powyzej slupków oznaczaja róznice statystycznie istotne wyznaczone za pomoca testu Tukey a dla p < 0.05. oznacza róznice statystycznie istotna pomiedzy wyciagami z korzeni slonecznika a kombinacja kontrolna wyznaczona za pomoca testu t- Studenta dla p < 0.05. Wartosci w slupkach reprezentuja zakres kielkowania w stosowanych stezeniach wyciagów. Fig. 2. Germination of winter wheat (Triticum aestivum L.) cv. Zyta in presence of allelochemicals contained in aqueous extracts from roots of 11 cultivars of sunflower (Helianthus annuus L.). Values in bars illustrate range in germination for all used concentrations. Rys. 2. Kielkowanie pszenicy ozimej (Triticum aestivum L.) odm. Zyta w obecnosci wodnych wyciagów korzeni 11 odmian slonecznika zwyczajnego (Helianthus annuus L.). Wartosci w slupkach reprezentuja zakres kielkowania w stosowanych stezeniach wyciagów. Fig. 3. Values of Pipper Index for white mustard (Sinapis alba L.) cv. Nakielska in presence of allelochemicals contained in aqueous extracts from roots of 11 cultivars of sunflower (Helianthus annuus L.). Bars marked by different letters differ significantly according to HSD Tukey test at p < 0.05. indicate significant difference between given concentration and control as estimated by the t-student s test at p < 0.05. Values in bars show range in time of germination for used extract concentrations. 11

Rys. 3. Czas kielkowania gorczycy bialej (Sinapis alba L.) odm. Nakielska w obecnosci wyciagów z korzeni 11 odmian slonecznika zwyczajnego (Helianthus annuus L.). Rózne litery powyzej slupków wskazuja na statystycznie istotne róznice pomiedzy stezeniami, wyznaczone za pomoca testu Tukey a dla p < 0.05. oznaczaja róznice statystycznie istotne pomiedzy wyciagami, a kombinacja kontrolna wyznaczona za pomoca testu t- Studenta dla p < 0.05. Wartosci w slupkach reprezentuja zakres czasu kielkowania w stosowanych stezeniach wyciagów. Fig. 4. Values of Pipper Index for winter wheat (Triticum aestivum L.) cv. Zyta seeds in presence of allelochemicals contained in aqueous extracts from roots of 11 cultivars of sunflower (Helianthus annuus L.). Bars marked by different letters differ significantly according to HSD Tukey test at p < 0.05. indicate significant difference between given concentration and control as estimated by the t-student s test at p < 0.05. Values in bars show range in time of germination for used extract concentrations. Rys. 4. Czas kielkowania pszenicy ozimej (Triticum aestivum L.) odmiana Zyta wyciagów z korzeni 11 odmian slonecznika zwyczajnego (Helianthus annuus L.). Rózne litery powyzej slupków wskazuja na statystycznie istotne róznice pomiedzy stezeniami, wyznaczone za pomoca testu Tukey a dla p < 0.05. oznaczaja róznice statystycznie istotne pomiedzy wyciagami, a kombinacja kontrolna wyznaczona za pomoca testu t- Studenta dla p < 0.05. Wartosci w slupkach reprezentuja zakres czasu kielkowania w stosowanych stezeniach wyciagów. Fig. 5. Fresh weight of white mustard (Sinapis alba L.) cv. Nakielska plants grown for 28 days in the soil previously used for cultivation of 4 cvs. of sunflower (Helianthus annuus L.) and deprived of any visible sunflower roots. Data are mean ± SE, n=10. Rys. 5. Swieza masa roslin gorczycy bialej (Sinapis alba L.) odm. Nakielska rosnacych przez 28 dni w glebie, w której wczesniej uprawiano rosliny 4 odmiany slonecznika zwyczajnego (Helianthus annuus L.) po usunieciu korzeni slonecznika. Dane przedstawiaja srednie z 10 powtórzen ± SE. 12

Fig. 6. Length of white mustard (Sinapis alba L.) cv. Nakielska seedlings as influenced by allelochemicals exuded by seedlings of 2 cvs. of sunflower (Helianthus annuus L.). Values above columns are expressed as % of control. Data are mean ± SE, n = 8 Rys. 6. Dlugosc siewek gorczycy bialej (Sinapis alba L.) odm. Nakielska rozwijajacych sie w obecnosci substancji allelopatycznych uwalnianych przez siewki 2 odmian slonecznika zwyczajnego (Helianthus annuus L.). Wartosci powyzej kolumn wyrazaja procent kontroli. Dane przedstawiaja srednie z 8 powtórzen ± SE. 13

100 a ab c bc bc bc c c c c c Germination (%) Zdolnosc kielkowamia (%) 80 60 40 20 96.5-99.5 83.3-90.5 88.8-94.3 91.8-95.3 91.8-96.0 90.8-96.9 92.3-96.8 93.0-96.8 95.3-96.9 95.3-96.9 94.5-96.9 95.3-97.8 0 control Pardisol Printasol Wielkopolski Pastewny Optisol Lech Polizie 57859 Albena Giganta Ogrodowy 14

100 Germination (%) Zdolnosc kielkowania (% 80 60 40 20 99.3-100 96.0-99.0 95.7-100 96.0-99.0 96.0-99.7 97.0-99.3 98.7-99.7 99.0-99.3 99.0-99.7 99.3-993 99.0-99.7 99.0-100 0 control Lech Albena Pardisol Pastewny Optisol Polizie Printasol 57958 Wielkopolski Ogrodowy Giganta 15

Pipper Index (days) Wspólczynnik Pippera (dni) 3,5 3,0 2,5 2,0 1,5 1,0 0,5 1.98-2.42 a 2.03-2.30 ab 2.11-2.71 bc 2.37-2.89 cd 2.09-3.19 cd 2.67-2.85 de 2.63-3.33 ef 2.06-4.23 ef 2.45-3.84 ef 2.56-3.77 f 2.81-3.73 f 2.92-3.64 0,0 control Albena Pastewny Polizie Printasol Ogrodowy Giganta Pardisol 57859 Optisol Lech Wielkopolski 16

Pipper Index (days) Wspólczynnik Pippera (dni) 2 1,5 1 0,5 1.7 a 1.44-1.67 ab ab bc cd 1.41-1.70 1.49-1.72 1.62-1.69 1.69-1.78 d 1.76-1.86 d 1.74-1.87 d 1.78-18.9 d 1.78-1.97 e 1.73-2.03 e 1.87-2.04 0 control Optisol Pardisol Lech Ogrodowy Printasol Albena Giganta Pastewny Polizie Wielkopolski breeding line 17

2 HSD 0,05 = 0,65 (above ground organs) NIR 0,05 = 0,65 (czesc nadziemna) HSD 0,05 = 0,19 (roots) NIR 0,05 = 0,19 (korzenie) 1,5 FW g plant -1 SM g roslina -1 1 0,5 above ground organs czesc nadziemna roots korzenie 0 control Albena Wielkopolski Pardisol Pastewny kontrola 18

6 whole seedling; cala siewka 100% 5 Length (cm) Dlugosc (cm) 4 3 2 1 hypocotyle hypokotyl 100% 21,8% 48,9% 100% root korzen 52,1% 59,2% 35,8% 53,7% 0 control exudates cv.ogrodowy exudates cv.lech kontrola wydzieliny odmiana Ogrodowy wydzieliny odmiana Lech 19

Table 1. Values of Pipper Index (days) for white mustard (Sinapis alba L.) cv. Nakielska as influenced by allelochemicals contained in aqueous root extracts of 11 cultivars of sunflower (Helianthus annuus L.). Bold refers to highest and lowest values. Values in lines (1) followed by different letter differ significantly as evaluated by Tukey test at p < 0.05. Tabela 1. Wpólczynnik Pipera (dni) dla gorczycy bialej (Sinapis alba L.) odm. Nakielska kielkujacej w obecnosci substancji allelopatycznych zawartych w wodnych wyciagach korzeni 11 odmian slonecznika zwyczajnego (Helianthus annuus L.). Pogrubienia wskazuja najwyzsza i najnizsza wartosc. Rózne litery przy wartosciach w wierszach (1) oznaczaja róznice statystycznie istotne wyznaczone za pomoca testu Tukey a na poziomie istotnosci p < 0.05. Cultivar Odmiana H 2 O (control) Extract concentration (DM % w/v) Stezenie wyciagu (SM % v/w) (kontrola) 2,5 5,0 7,5 10,0 Albena 1,98 a 2,03 ab (1) 2,13 ab 2,24 b 2,30 b Pastewny 1,98 a 2,11 ab 2,42 b 2,49 bc 2,71 c Polizie 1,98 a 2,37 b 2,32 b 2,55 b 2,89 b Printasol 1,98 a 2,09 a 2,51 ab 3,08 b 3,19 b Ogrodowy 2,42 a 2,67 b 2,76 b 2,77 b 2,85 b Giganta 2,42 a 2,63 a 2,65 a 3,29 b 3,33 b Pardisol 1,98 a 2,06 ab 2,56 b 3,48 c 4,23 d 57859 1,98 a 2,45 ab 2,49 ab 3,80 b 3,84 b Optisol 2,42 a 2,56 ab 3,08 bc 3,40 cd 3,77 d Lech 2,42 a 2,81 a 2,90 a 3,59 b 3,73 b Wielkopolski 2,42 a 2,92 ab 3,22 bc 3,32 bc 3,64 c Average Srednia 2,18 2,53 2,66 3,08 3,32 20

Table 2. Values of Pipper Index (days) for winter wheat (Triticum aestivum L.) cv. Zyta as influenced by allelochemicals contained in aqueous root extracts of 11 cultivars of sunflower (Helianthus annuus L.). Bold refers to highest and lowest values. Values in lines (1) followed by different letter differ significantly as evaluated by Tukey test at p < 0.05.. Tabela 2. Wspólczynnik Pipera (dni) dla pszenicy ozimej (Triticum aestivum L.) odm. Zyta kielkujacej w obecnosci substancji allelopatycznych zawartych w wodnych wyciagach z korzeni 11 odmian slonecznika zwyczajnego (Helianthus annuus L.) Pogrubienia wskazuja najwyzsza i najnizsza wartosc. Rózne litery przy wartosciach w wierszach (1) oznaczaja róznice statystycznie istotne wyznaczone za pomoca testu Tukey a na poziomie istotnosci p < 0.05.. Cultivar Odmiana H 2 O (control) Extract concentration (DM % w/v) Stezenie wyciagu (SM % w/v) (kontrola 2,5 5,0 7,5 10,0 Optisol 1,71 b 1,44 a (1) 1,46 a 1,52 ab 1,67 b Pardisol 1,71 b 1,41 a 1,63 b 1,64 b 1,70 b Lech 1,71 b 1,49 a 1,59 ab 1,66 ab 1,72 b Ogrodowy 1,71 a 1,62 a 1,64 a 1,67 a 1,69 a Printasol 1,71 a 1,69 a 1,69 a 1,78 a 1,78 a Albena 1,71 a 1,76 a 1,79 a 1,80 a 1,86 a Giganta 1,71 a 1,74 a 1,74 a 1,88 a 1,87 a Pastewny 1,71 a 1,78 a 1,84 ab 1,89 b 1,89 b Polizie 1,71 a 1,78 a 1,84 ab 1,89 ab 1,97 b Wielkopolski 1,71 a 1,73 a 1,97 b 1,98 b 2,03 c 57958 1,71 a 1,87 b 2,00 bc 2,02 c 2,04 c Average Srednia 1,71 1,66 1,80 1,79 1,84 21

Table 3. Changes in fresh matter of shoot (S) and roots (R) and in the R:S ratio in white mustard (Sinapis alba L.) cv. Nakielska plants grown for 28 days in the soil previously used for sunflower cultivation and deprived of any visible roots. Data are mean ± SE, n = 8. Tabela 3. Swieza masa czesci nadziemnej i korzeni roslin gorczycy bialej (Sinapis alba L.) odm. Nakielska oraz stosunek korzen:lodyga rosnacych przez 28 dni w glebie, w której uprzednio uprawiano rosliny 4-ch odmian slonecznika. Dane przedstawiaja srednie z 8 powtórzen ± SE. Combination/ Cultivar Kombinacja/ Odmiana H 2 O (Control) Kontrola % of control; % kontroli whole seedling above ground cala siewka organs czesc nadziemna roots korzenie Root : Shoot Korzen : Ped 100 100 100 0,31 Albena 17,5 8,0 48,1 1,86 Wielkopolski 18,9 6,3 59,3 2,91 Pardisol 16,2 9,2 38,5 1,31 Pastewny 14,9 6,3 42,6 2,09 22