Allelopathic potential of invasive alien species tree of heaven (Ailanthus altissima (Mill.) Swingle)

Alelopatija je pojam koji opisuje biokemijske reakcije između biljaka uslijed lučenja alelokemikalija. Mnoge invazivne biljne vrste imaju visok alelopatski potencijal. Invazivna alohtona vrsta pajasen (Ailanthus altissima (Mill.) Swingle) je jedna od najčešće istraživanih vrsta radi izolacije i pronalaženja alelokemikalija s herbicidnim učinkom. Cilj ove disertacije je odrediti alelopatski učinak vodene otopine korijena pajasena i iste količine izoliranog ailantona na klijavost i početni rast i razvoj dvije korovne vrste, oštrodlakavog šćira (Amaranthus retroflexus L.) i muhara (Setaria pumila (Poir.) Schult.) te kultiviranu vrstu kukuruz (Zea mays L.). Vodena otopina korijena pajasena pripremljena je od mladih izbojaka. Identifikacija i kvantifikacija ailantona iz vodene otopine korijena pajasena određena je tekućinskom kromatografijom kojom je određena koncentracija ailantona od 0,48 mg/ml. Potom je diklormetanom izoliran ailanton iz vodene otopine korijena pajasena. Od pripremljenih vodenih otopina izoliranog ailantona i korijena pajasena pripremljena je koncentrirana otopina i pet razrjeđenja destiliranom vodom u omjeru 3/2; 1/2; 1/4; 1/8 i 1/16 koja su ekvivalent određenoj koncentraciji ailantona u vodenoj otopini korijena pajasena, koncentrat s 0,48 mg/ml ailantona i pet razrjeđenja s 0,32; 0,24; 0,12; 0,06 i 0,03 mg/ml ailantona. Pripremljena razrjeđenja vodene otopine izoliranog ailantona i vodene otopine korijena primijenjena su na sjeme (pre-em) i na list (post-em) kad su test-biljne vrste muhar i kukuruz bile u stadiju rasta 3 lista (BBCH 13), a oštrodlakavi šćir u stadiju rasta 2 - 4 lista (BBCH 12-14). Dokazano je da se istraživane test-biljne vrste razlikuju u osjetljivosti prema vodenoj otopini izoliranog ailantona i vodenoj otopini korijena pajasena. Primjenom vodenih ekstrakata na sjeme muhara dokazan je inhibirajući alelopatski učinak na sva istraživana svojstva (klijavost, duljina korijena klice i duljina izdanka klice), a najznačajnija povezanost između alelopatskog učinka i istraživanih koncentracija ailantona dokazana je kod klijavosti kad su test- biljne vrste tretirane s vodenom otopinom korijena pajasena. Obje istraživane vodene otopine su inhibirale duljinu korijena i izdanka klice oštrodlakavog šćira. Najznačajnija povezanost alelopatskog učinka i koncentracija ailantona kod oštrodlakavog šćira dokazana je za klijavost kad je tretiranje obavljeno s vodenom otopinom korijena pajasena. Za razliku od korovnih vrsta, kod kukuruza je dokazano inhibitorno djelovanje samo na duljinu korijena klice uz statistički značajnu povezanost istraživanih koncentracija i alelopatskog učinka kad je tretiranje obavljeno s obje vodene otopine. Nasuprot inhibiciji, kod kukuruza je utvrđena i stimulacija kod djelovanja na duljinu izdanka klice kad je tretiranje obavljeno s vodenom otopinom korijena pajasena. Primjenom na list, obje istraživane vodene otopine izoliranog ailantona i korijena pajasena iskazale su inhibirajuće djelovanje na sve tri istraživane test-biljne vrste. Jedino je koncentracija od 0,48 mg/ml ailantona iz vodene otopine izoliranog ailantona povećala masu nadzemnog dijela kukuruza, ali povećanje nije bilo statistički značajno. Statistički najznačajnija povezanost intenziteta alelopatskog učinka i istraživanih koncentracija ailantona dokazana je kod oštrodlakavog šćira kad je tretiranje obavljeno s obje vodene otopine. Kod muhara nije dokazana statistički značajna razlika u povezanosti između alelopatskog učinka i istraživanih koncentracija ailantona iz obje vodene otopine. Nasuprot tome, kod kukuruza je statistički značajna povezanost alelopatskog učinka i koncentracija ailantona dokazana samo kad je tretiranje obavljeno s vodenom otopinom izoliranog ailantona. Kod muhara i oštrodlakavog šćira vodena otopina korijena pajasena primijenjena na sjeme iskazala je jače djelovanje u usporedbi s vodenom otopinom izoliranog ailantona kod svih istraživanih svojstava kod kojih je određena statistički značajna razlika. Nasuprot tome, kod kukuruza je u istom roku primjene, statistički značajnije djelovanje kod svih istraživanih svojstava dokazano kad je tretiranje obavljeno s vodenom otopinom izoliranog ailantona. Primjenom na list vodena otopina korijena pajasena iskazala je jače djelovanje na nadzemnu masu oštrodlakavog šćira i kukuruza, a vodena otopina izoliranog ailantona na muhara. Vodena otopina korijena pajasena iskazala je jače djelovanje u usporedbi s vodenom otopinom izoliranog ailantona kod većine istraživanih svojstava što upućuje na to da ailanton nije jedina alelokemikalija u korijenu pajasena. Iz određenih EC50 vrijednosti dokazano je da je oštrodlakavi šćir najosjetljivija, a kukuruz najtolerantnija test-biljna vrsta.The overuse of synthetic herbicides for weed control over the last five decades has resulted in growing public concern over their impacts upon human health, the environment, and the evolution of herbicide resistant weeds. Natural compounds from plants offer excellent potential for new herbicidal solutions, or lead compounds for new herbicides. Allelopathy is the common name for biochemical reaction between plants as a result of allelochemical secretion. Many invasive plant species have high allelopathic potential. Invasive alien species tree of heaven (Ailanthus altissima (Mill.) Swingle) is one of the most frequently investigated species for the purpose of isolating and finding allelochemicals with herbicidal effect. The aim of this dissertation was to determine the allelopathic effect of the aqueous solution of tree of heaven‘s root and the same amount of isolated ailanthone on germination and the initial growth and development of two weed species, pigweed (Amaranthus retroflexus L.) and yellow foxtail (Setaria pumila (Poir.) Schult.), and cultivated plant species maize (Zea mays L.). The young shouts with roots of tree of heaven were collected before flowering in late spring – early summer. Freshly collected roots were cut into small pieces (size 0.5-1 cm) and grained. In one litter of distilled water 250 g of grained plant material was soaked into at room temperature for 24 hours. After 24 hours, plant material was removed and extracts were filtered through filter paper (wrinkled 21/N, Munktel & Filtrak). Identification and quantification of ailanthone from root aqueous solution was made on HPLC. Confirmation of identity and quantification of ailanthone from the solution was determined by linear regression based on the calibration curve of the standard solution of the ailanthone (purity > 98 %). In tree of heaven’s root aqueous solution the concentration of 0.48 mg/ml of ailanthone was determined. After identification and quantification, isolation of ailanthone from one half of the root aqueous solution was performed with dichloromethane. The extraction of ailanthone was carried out three times in the separating funnel and the sample was further purified with sodium sulphate. The sample was then evaporated in rotary evaporator at 50 °C to a dry residue. Six dilutions of the root aqueous solution (1; 3/2; 1/2; 1/4; 1/8 and 1/16) and six dilutions of the ailanthone aqueous solution which were equivalent to the determinate concentration (0.48; 0.32; 0.24; 0.12; 0.06 and 0.03 mg/ml of ailanthone) were prepared. Experiments were carried out in the Weed Science laboratory at the Croatian agriculture and food agency - Centre for plant protection. The first part of the research that relates to the pre-emergence effect of the aqueous solutions on the test species was carried out in Petri dishes. In each sterilized Petri dish, 25 seeds of yellow foxtail, pigweed and maize were placed on two filter paper layers in four replicates. Before placing seeds, 4 ml of aqueous solution for yellow foxtail and pigweed, and 8 ml of aqueous solution for maize were added per Petri dish. For the control treatment, distilled water was used. Petri dishes were placed in darkness at 25 - 27 °C and relative humidity 70 % in a climate chamber. Percentage of germination, radicle length and shoot length were determined for each variant. All investigated attributes were determined at the same time for each test-species. Pigweed germination, radicle length and shoot length were measured 5 days after sowing, yellow foxtail was measured 7 days after sowing and maize was measured 6 days after sowing. It has been found that the test plants of studied species differ in the sensitivity to the aqueous solution of isolated ailanthone and the aqueous solution of the tree of heaven’s root. By applying aqueous solutions to the seeds of yellow foxtail, inhibitory allelopathic effects for all the investigated attributes (germination, radicle length and shoot length) were determined. The most significant correlation between the allelopathic effect on yellow foxtail and the investigated concentrations of ailanthone were determined for the germination in the treatment with the root aqueous solution. The both investigated aqueous solutions also inhibited the radicle length and shoot length. The most significant correlation between the allelopathic effect and the investigated concentrations of ailanton was also determined for germination in the treatment with the root aqueous solution. Unlike weed species, the effect of the investigated aqueous solutions inhibited only the radicle length of maize with a significant correlation between the investigated ailanthone concentrations in the treatments with both aqueous solutions. In contrast to the inhibition, in the case of corn, stimulation was found in the effect for the shoot length in the treatment with aqueous root solution. The second part of the research that relates to the post-emergence effect of the aqueous solutions on test - species was carried out by breeding 10 plants of yellow foxtail, pigweed and maize per repetition in containers. The containers were placed in a climate chamber. The experiment was set up in three repetitions. The application of all investigated concentrations of aqueous solutions and distilled water as control was made when the test- species reached the development stage of two to four leaves (BBCH 12 - 14). The allelopathic effect of the investigated solutions on the mass of the fresh plant mass of test - species was determined 16 days after application. Both investigated aqueous solutions of isolated ailanthone and tree of heaven’s roots showed inhibitory effect on all three studied test - plant species. Only a concentration of 0,48 mg/ml of ailanthone from aqueous solution of isolated ailanthone increased the above - ground mass of the corn, but it was not statistically significant. The highest correlation between the allelopathic effect and the investigated concentrations of ailanthone was found for the pigweed on treatments with both aqueous solutions. The statistical significant correlation between the effect and the investigated concentrations of ailanthone from both aqueous solutions was not established for the yellow foxtail. In contrast, in maize, statistically significant correlation was established only in the treatment with aqueous solution of isolated ailanthone. In the case of yellow foxtail and pigweed, in the pre-emergence application, the aqueous solution of the tree of heaven’s root showed a stronger effect compared to the aqueous solution of the isolated ailanthone for the all investigated attributes in which a statistically significant difference was established. As for maize, in the same application period, the aqueous solution of isolated ailanthone showed a stronger effect for all investigated attributes compared to the aqueous solution of the tree of heaven’s root. In the postemergence application, the aqueous solution of the root showed a stronger effect on the above - ground mass of the pigweed and maize, and an aqueous solution of isolated ailanthone on yellow foxtail. The aqueous solution of tree of heaven’s roots generally showed a stronger effect compared to the aqueous solution of the isolated ailanthone for the most investigated attributes, suggesting that the ailanthone is not the only allelochemical in the root of tree of heavens, but was certainly the most potent one. The most sensitive test-species was a pigweed, and the most tolerant maize