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Cilj doktorske disertacije je bil: i) preučiti toleranco in tolerančne mehanizme treh temnih septiranih (TSE) endofitov, izolata Cad#148 in Cad#149 iz rodu Cadophora in izolata Leo#146 iz rodu Leohumicola na visoke koncentracije Cd, Zn in Pb v Melin-Norkrans (MNM) gojišču ter ii) nadalje v lončnem poskusu preučiti njihovo primernost za remediacijo onesnaženega substrata z vrbo ivo (Salix caprea) v kombinaciji z različno vrsto podrasti. Izolata iz rodu Cadophora sta v miceliju tvorila 1,8-dihidroksinaftalen (DHN)-tip melanina, pri čemer je bila sinteza večja v TSE izolatu Cad#148. Izolat Leo#146 pa je bil nemeliniziran. Občutljivost izolatov na kovine je bila največja na Cd, sledita Zn in Pb. Melanizirana izolata sta bila v splošnem bolj tolerantna na kovine, zaradi manjše akumulacije kovin v miceliju in manjšega oksidativnega stresa. Sinteza organskih kislin je bila povečana v prisotnosti Cd in Zn stresa, pri čemer je bila vrsta induciranih organskih kislin za izolat specifina. Sinteza melanina se je v melaniziranih izolatih zmanjševala z naraščajočim kovinskim stresom. Kljub temu smo z uporabo inhibitorja melaninske sinteze v prisotnosti Cd stresa dokazali, da ima melanin pomembno vlogo pri toleranci na Cd najbolj melaniziranega TSE izolata Cad#148. V nadaljnjem lončnem poskusu smo preučili vpliv inokulacije z izbranimi TSE izolati in različne vrste podrasti (naravne, zasejane) na fiziologijo in akumulacijo kovin v vrbi ivi, ukoreninjenih v nesterilnem onesnaženem substratu. TSE izolata Leo#146 in Cad#148 sta najuspešnejše inficirala korenine vrbe ive in pozitivno vplivala na rast in sintezo skupnih klorofilov in karotenoidov v listih vrb. Inokulacija vrb s TSE izolatom Leo#146 je v kombinaciji s podrastjo pisane vilovine zmanjšala translokacijo Cd in Zn iz korenin v liste vrb in posledično zmanjšala akumulacijo Cd in Zn v listih. Poleg tega je inokulacija potaknjencev vrbe ive s TSE izolatoma Leo#146 in Cad#148 v kombinaciji s podrastjo Rožnik zmanjšala translokacijo Pb iz korenin v liste. TSE izolat Leo#146 je bil v kombinaciji s podrastjo pisana vilovina izbran za najprimernejši izolat za fitoremediacijo onesnaženega substrata z vrbo ivo. The aim of the doctoral dissertation was: i) to investigate the tolerance and tolerance mechanisms of three dark septate endophyte (DSE) isolates, Cad#148 and Cad#149 from the genus Cadophora and isolate Leo#146 from the genus Leohumicola to high concentrations of Cd, Zn and Pb in Melin-Norkrans (MNM), and ii) to evaluate their suitability for remediation of contaminated substrate with goat willow (Salix caprea) in combination with different types of undergrowth. Isolates from the genus Cadophora formed 1,8-dihydroxynaphthalene (DHN) melanin in mycelium, with isolate Cad#148 showing higher synthesis. Isolate Leo#146 was not melanized. The sensitivity of isolates to metals was highest for Cd, followed by Zn and Pb, with the melanized isolates showing higher tolerance due to lower accumulation of metals in the mycelium and lower oxidative stress. Organic acid synthesis was increased in the presence of Cd and Zn stress, although the type of organic acids induced was isolate-specific. Melanin synthesis in melanized isolates decreased with increasing metal stress. However, by using a melanin synthesis inhibitor in the presence of Cd stress, we demonstrated that melanin plays an important role in the tolerance of the most melanized DSE isolate Cad#148 to Cd. Furthermore, in a pot experiment, we investigated the influence of inoculation with selected DSE isolates and different types of undergrowth (natural, sown) on the physiology and accumulation of metals in willow rooted in non-sterile contaminated substrate. DSE isolates Leo#146 and Cad #148 were most successful in infecting willow roots and had positive affect on growth and synthesis of total chlorophyll and carotenoid pigments in willow leaves. Inoculation of willows with the DSE isolates Leo#146 in combination with undergrowth blue moor grass reduced the transfer of Cd and Zn from roots to leaves and consequently accumulation of Cd and Zn in leaves. In addition, inoculation of willow cuttings with Leo#146 and Cad#148 in combination with undergrowth Rožnik reduced the translocation of Pb from roots to leaves. The DSE isolate Leo#146 in combination with the undergrowth of blue moor grass was selected as the most suitable isolate for phytoremediation of contaminated substrate with willow. |
