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Täistekst (6.017Mb) EESTI MAAÜLIKOOL Metsandus- ja maaehitusinstituut Kati Küngas VALITUD ENDOFÜÜTSETE SEENTE JA ARMILLARIA spp. ANTAGONISM IN VITRO Bakalaureusetöö loodusvarade kasutamise ja kaitse erialal Juhendaja dotsent Bellis Kullman Tartu 2014 Lõputöö koostasin iseseisvalt. Kõigile töös kasutatud teiste autorite töödele ja andme- allikatele on viidatud. Kinnitan, et annan oma intellektuaalomandi varalised õigused lõputöö suhtes üle Eesti Maaülikoolile. ........................................................................ kuupäev/ nimi/ allkiri/ Tunnistan lõputöö kaitsevalmiks. Juhendaja: …………………………………………........ kuupäev/ nimi/ allkiri Eesti Maaülikool Bakalaureusetöö lühikokkuvõte Kreutzwaldi 1, Tartu 51014 Autor:Kati Küngas Õppekava:Loodusvarade kasutamine ja kaitse Pealkiri: Valitud endofüütsete seente ja Armillaria spp. antagonism in vitro Lehekülgi: 89 Jooniseid: 9 Tabeleid: 6 Lisasid: 14 Osakond: PKI mükoloogia Uurimisvaldkond: mükoloogia Juhendaja: dotsent Bellis Kullman Kaitsmiskoht ja aasta: Tartu, 2014 Külmaseene ( Armillaria) liigid on levinud üle kogu maailma, nakatades erinevaid peremeestaimi ja põhjustades metsades suurt majanduslikku kahju. Endofüütsed seened elavad peremeestaime sees kahjustusi põhjustamata. Mitmed uurimused on näidanud, et endofüüdid on peremeestaimele kasulikud stimuleerides kasvu ning kaitstes haiguste eest (Gao et al . 2010). Töö eesmärgiks oli külmaseente ja endofüütide vaheliste kaksikkultuurtestide (dual culture tests ) abil uurida endofüütide antagonismi külmaseente suhtes. Töös kasutati 9 erinevat endofüütset seent: Annulohypoxylon multiforme, Aureobasidium pullulans, Epicoccum nigrum, Hypoxylon howeanum, Phoma multirostrata, Phoma var . verbascicola, Fusarium culmorum, Lewia infectoria, Cladosporium tenuissimum ja kolm külmaseene liiki: põhja- külmaseen ( Armillaria borealis), tutt-külmaseen (Armillaria cepistipes), tõmmu külmaseen (Armillaria ostoyae). Uurimuses antakse ülevaade antud katses kasutatavatest endofüütidest biotõrjes ja külmaseentest kui patogeenidest. Töös arvutatakse antagonismiprotsendid iga patogeeni suhtes. Katse läbiviimisel kasutatakse Kusari et al. (2013) metoodikat. Töö tulemusena antakse ülevaade mõnede endofüütide kasutatavusest külmaseente biotõrjes. Kõik valitud endofüütsed seened pärssisid külmaseente kasvu in vitro 2% MEA söötmel. Kõige rohkem pärssis tutt-külmaseene kasvu F. culmorum , tõmmu külmaseene kasvu A. multiforme ja põhja-külmaseene kasvu P. poolensis var . verbascicola (antagonismiprotsent vastavalt 67 ±6,8, 77 ±1,5 ja 79 ±4,2). A. multiforme pärssis nii põhja- külmaseene kui ka tõmmu külmaseene kasvu kõikides katses üle 70%. Antud töös parimateks külmaseente tõrjujateks olid A. multiforme , P. poolensis var . verbascicola ja E. nigrum. Endofüütset seent A. multiforme võib soovitada ka teiste külmaseente liikide, näiteks A. mellea vastu , kes põhjustab üle maailma majanduslikku kahju nii põllumajanduses kui ka metsanduses. Tuvastamaks parimaid endofüütseid seenetüvesid külmaseente biotõrjeks, tuleks edaspidises töös katsetada puude nakatamist endofüütidega looduses ja kasutada spetsiifilisi bioloogiliselt aktiivseid ühendeid, mida endofüüdid oma elutegevuse käigus toodavad. Märksõnad: endofüüdid, Armillaria spp., antagonism, kaksikkultuurtest, biotõrje Estonian University of Life Sciences Abstract of Bachelor’s Thesis Kreutzwaldi 1, Tartu 51014 Author: Kati Küngas Specialty: Managment of natural resources Title: Antagonistic effect of selected endophytic fungi against Armillaria spp. in vitro Pages: 89 Figures: 9 Tables: 6 Appendixes: 14 Department: PKI mycology Field of research: mycology Supervisors: assistant professor Bellis Kullman Place and date: Tartu, 2014 Armillaria spp. is widespread around the world. The host range of Armillaria spp. is wide, it can infect many host species and causes serious economic loss to forests. Endophytic fungi are microbes that live in the healthy tissue of the host plant causing no damage. There are many studies showing that endophytes are beneficial to their hosts stimulating growth and providing protection from diseases (Gao et al .2011). The aim of this study was to perform dual culture tests between Armillaria spp. and endophytic fungi to evaluate the antagonistic effect between these species. Nine different endophytic fungi were used in this study: Annulohypoxylon multiforme, Aureobasidium pullulans, Epicoccum nigrum, Hypoxylon howeanum, Phoma multirostrata, Phoma var. verbascicola, Fusarium culmorum, Lewia infectoria, Cladosporium tenuissimum and three Armillaria spp. species: A. borealis, A. cepistipes, A. ostoyae. This study gives an overview of endophytes currently used as biocontrol agents in the world and of Armillaria spp. as pathogens. Percentages of antagonism against each pathogen were calculated in the assay. The methodology of Kusari et al. (2013) was used in the dual culture tests. The results of this study give an overview of the potential of some endophytic fungi as a source of biocontrol agents against Armillaria spp. All selected endophytic fungi inhibited the growth of Armillaria spp. in vitro using MEA 2% media. F. culmorum inhibited the growth of A. cepistipes the most (percentage of antagonism 67 ±6.8). A. multiforme inhibited the growth of A. ostoyae the most (percentage of antagonism 77 ±1.5). P. poolensis var. verbascicola inhibited the growth of A. borealis the most (percentage of antagonism 79 ±4.2). In each test A. multiforme inhibited the growth of A. ostoyae and A. borealis over 70%. A. multiforme, P. poolensis var . verbascicola and E. nigrum are considered, in this study, the best biocontrol agents against Armillaria spp. Endophytic fungus A. multiforme should also be tested against other Armillaria species such as A. mellea that causes economic loss to forests and the agricultural sector on a global scale. In the future, further studies should be conducted with endophyte infected trees and also into disease control by specific biologically active compounds produced by endophytes in order to select the most fitting species for biological control against Armillaria spp. Keywords: Endophytes, Armillaria spp., antagonism, dual culture tests, biocontrol SISUKORD Sissejuhatus ............................................................................................................................ 7 1. Kirjanduse ülevaade ........................................................................................................... 9 1.1. Endofüütsete seente kasutatavus biotõrjes .................................................................. 9 1.2. Valitud endofüütsed seened ....................................................................................... 10 1.2.1. Annulohypoxylon multiforme ja Hypoxylon howeanum ..................................... 10 1.2.2. Fusarium culmorum ............................................................................................ 11 1.2.3. Aureobasidium pullulans .................................................................................... 12 1.2.4. Caldosporium tenuissimum ................................................................................ 12 1.2.5. Phoma multirosrtata ja Phoma poolensis. var . verbascicola ............................. 13 1.2.6. Epicoccum nigrum .............................................................................................. 14 1.2.7. Lewia infectoria .................................................................................................. 15 1.3. Perekond külmaseen ( Armillaria ) ............................................................................. 16 1.3.1. Külmaseene keemiline ja bioloogiline tõrje ....................................................... 19 1.3.2. Valitud külmaseene liigid ................................................................................... 20 1.3.2.1. Põhja- külmaseen ( Armillaria borealis ) ja tutt-külmaseen ( Armillaria cepistipes ) ................................................................................................................. 20 1.3.2.2. Tõmmu külmaseen ( Armillaria ostoyae ) .................................................... 20 2. Materjal ............................................................................................................................ 22 3. Metoodika ........................................................................................................................ 24 4. Tulemused ........................................................................................................................ 27 4.1. Endofüütide antagonistlik toime põhja-külmaseenele ( Armillaria borealis ) ............ 28 4.1.1. Liigi Hypoxylon howeanum toime ...................................................................... 30 4.1.2. Liigi Fusarium culmorum toime ......................................................................... 30 4.1.3. Liigi Aureubasidium pullulans toime ................................................................. 30 4.1.4. Liigi Cladosporium tenuisimmum toime ............................................................ 31 4.1.5. Liigi Phoma poolensis var. verbascicola toime.................................................. 31 4.1.6. Liigi Phoma multirostrata toime ........................................................................ 31 4.1.7.Liigi Epicoccum nigrum toime ............................................................................ 32 4.1.8. Liigi Annulohypoxylon multiforme toime ........................................................... 32 4.1.9.
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