Application of flow cytometry to measure genome and spore sizes of selected Basidiomycota groups

Voolutsütomeetriat kasutatakse valdavalt meditsiinis haigustekitajate ja rakutsükli uurimiseks. Seene eoste ja genoomi suuruse mõõtmiseks on varem kasutatud valgusmikroskoopiat. Eose suurus on oluline tunnus seente süstemaatikas, mida kasutatakse nii liikide piiritlemisel kui liikide määramisel (Parmasto E. ja Parmasto I. 1987). Genoomi suurus, tuuma DNA sisaldus ja polüploidsus on olulised parameetrid seente elurikkuse uurimisel. Uuringu eesmärk oli rakendada voolutsütomeetriat taelikute (Phellinus) ja servikute (Pleurotus) liikidel eose ja genoomi suuruse määramisel. Töötati välja uus metoodika ellipsoidsete seeneeoste mõõtmiseks. Eostest genoomis suuruse määramiseks lahendati mitmeid probleeme. Töötati välja uus metoodika eostest tuumade väljutamiseks sest selgus, et genoomi suurust eoste tugeva autofluoresentsi tõttu paksukestaliste eoste tuumadest mõõta ei saa. Seevastu selgus, et eoseid ja tuumi on lihtne grupeerida tänu autofluorestentsi olemasolule nende suuruse mõõtmiseks. Eose autofluoresentsi intensiivsus ise aga uue tunnusena võimaldab koos eose suurusega kasutades eritada gruppe, mis ainult eose pikkuse ja laiuse järgi ei eritu. Töö käigus eraldati intaktsed tuumad liikidel austerservik (Pleurotus ostreatus) ja kopsservik (P. pulmonarius). Uuringu käigus mõõdeti 136 seene eoste suurused ning 16 seene genoomi suurus. Tuuma suuruse ja DNA-sisalduse vahel on seos korrelatsioonikordajaga 0,75. Intaktsete tuumade mõõtmine väldib eoste autofluorestsentsist ja eosproovis mingi osa mitmiktuumadega eoste olemasolust tekkida võivat tegelikust suurema genoomi saamist. Genoomi suuruste suhteliseks määramiseks kasutati referentorganismi austerservik (Pleorotus ostreatus) TAAM126992 (genoomi suurusega 32 Mbp) kasutades selleks rakutsükli faasis G1 olevaid tuumi. Uuringu käigus välja töötatud läbivoolutsütomeetria metoodeid on võimalik kasutada seente süstemaatika jaoks eoste ja genoomi suuruse järgi liikide grupeerimiseks, liikide piiritlemiseks ja määramiseks. Voolutsütomeetria võimaldab kiiresti saada täpseid andmeid eosproovis olevate eoste mitmete parameetrite varieeruvuse kohta. Kasutades etalone saab suhtelistesed mõõdud konverteerida absoluutseteks ja teiste meetoditega saadud andmetega koos kasutadaFlow cytometry is mainly used in medicine to study pathogens and cell cycles. Fungal spore size is one of the most important characteristics in fungal taxonomy when studying species delimitation or determining species (Parmasto E. and Parmasto I. 1987). Previously, fungal spore and genome sizes were usually measured using light microscopy. Genome size, nuclear DNA content, and polyploidy are important if exploring fungal diversity. The aim of this study was to implement flow cytometry to measure spore and genome sizes of selected fungal groups of genus Phellinus and Pleurotus. A new methods for measuring elliptical spore sizes was developed for this. Due to strong autofluorescence of spores genome size was hard to measure for thick-walled spores and a new way to excrete nuclei of spores was therefore developed. In contrast, autofluorescence allowed for an easier way select groups of spore for further measuring. Using spore sizes and intensity of autofluorescence measured by flow cytometry allows for grouping specimen where differentiation based on spore length and width is difficult. Intact nuclei were isolated from Pleurotus ostreatus and P. pulmonarius spores. During this study, spores of 136 fungi and genome sizes of 16 fungi were measured. Correlation coefficient between DNA content and volume of the nucleus was 0,75. Measuring only intact single nucleus of spore excludes possibility to make mistakes by measuring two nuclei as one. To determine genome size reference organism (Pleorotus ostreatus) TAAM126992 (genome size 32 Mbp) cell cycle G1 phase spores were used. The study provides new methods for flow cytometry to measure spores size of elliptical spores and obtain intact nuclei for measure their DNA content. Using flow cytometry, large amounts of spores could be measured in minutes and using random sampling. Using two parameter analysis of spore sizes and intensity of autofluorescence reveal groups for fungal taxonomy to identify and delimit species

FungalTraits:A user-friendly traits database of fungi and fungus-like stramenopiles

The cryptic lifestyle of most fungi necessitates molecular identification of the guild in environmental studies. Over the past decades, rapid development and affordability of molecular tools have tremendously improved insights of the fungal diversity in all ecosystems and habitats. Yet, in spite of the progress of molecular methods, knowledge about functional properties of the fungal taxa is vague and interpretation of environmental studies in an ecologically meaningful manner remains challenging. In order to facilitate functional assignments and ecological interpretation of environmental studies we introduce a user friendly traits and character database FungalTraits operating at genus and species hypothesis levels. Combining the information from previous efforts such as FUNGuild and Fun(Fun) together with involvement of expert knowledge, we reannotated 10,210 and 151 fungal and Stramenopila genera, respectively. This resulted in a stand-alone spreadsheet dataset covering 17 lifestyle related traits of fungal and Stramenopila genera, designed for rapid functional assignments of environmental studies. In order to assign the trait states to fungal species hypotheses, the scientific community of experts manually categorised and assigned available trait information to 697,413 fungal ITS sequences. On the basis of those sequences we were able to summarise trait and host information into 92,623 fungal species hypotheses at 1% dissimilarity threshold

Antagonistic effect of selected endophytic fungi against Armillaria spp. in vitro

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 katsetes 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.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