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Estimation of fungal spore size using flow cytometry
Get PDFKäesoleva bakalaureusetöö eesmärgiks oli katsetada voolutsütomeetriat eoste
suurusparameetrite hindamiseks liudikulaadsete seente näitel nende süstemaatika tarbeks.
Võrreldi voolutsütomeetriat praegusel ajal seeneeoste mõõtmiseks kasutatava metoodikaga –
valgusmikroskoopiaga. Anti ülevaade voolutsütomeetri kasutusaladest nii maailmas kui Eestis
ning toodi välja mõlema head ja halvad küljed.
Töös kasutatud liudikulaadsete seente eoseid mõõdeti kahel viisil – voolutsütomeetri ja
valgusmikroskoobi abil. Eosproovid pärinevad Eesti Maaülikoolile kuuluvast sporoteegist.
Uue mõõtmismeetodi väljatöötamiseks valiti esindajad liudikulaadsete seltsi erinevatest
perekondadest. Kokku töötati 53 eksemplariga. Igast perekonnast püüti saada 5-7 esindajat.
Perekondi valiti kokku 24, milledest liigini määratud eksmplare oli 24. Arvukamalt olid
esindatud perekonnad Sarcoscypha (8), Peziza (6) ja Morchella (6).
Mõõtmiste tulemusel selgus, et mõõtes seeneeoseid voolutsütomeetriga ja
valgusmikroskoobiga saame eoste pikkuse laiuse mõõdud vägagi sarnased. Sama eosproovi
lineaarmõõdud olid valgusmikroskoobis 30% suuremad kui voolutsütomeetris, kuid pärast
paranduste sisseviimist oli tulemuste erinevus vaid 5% (s.o keskmine üle kogu mõõdetud
materjali).
Tööst selgub, et valgusmikroskoopiat saab asendada voolutsütomeetriaga ja kasutada
süstemaatikas esinevate probleemide uurimiseks. Eriti sobib ta sõsar- ja kompleksliikide
uurimiseks eoste ekvivalentdiameetri alusel. Ta sobib liigi struktuuri uurimiseks, et võrrelda
erinevas geograafilises piirkonnas kasvavaid seenepopulatsioone omavahel. Tulevikus võiks
uurida, kuidas mõjutab eose ornamentatsioon ja ebasümmeetrilisus voolutsütomeetriga
mõõtmisel mõõtmistulemust. Voolutsütomeetriat oleks oluline kasutada juhtudel, kui
mõõdetakse allergiaid põhjustavate seeneeoste konsentratsiooni õhus.The aim of this study was to test flow cytometry for estimation of fungal spore size for fungal
systematics. Flow cytometry was compared with light microscopy which is traditionaly used.
An overview of flow cytometry usage in the World and in Estonia was given. Flow cytometry
and light microscopy merits and demerits were brought out.
The fungal spores of Pezizales were measured in two ways –by flow cytometer and by light
microscope. The spore samples used in this study came from fungarium of the mycology
department of the University of Life Sciences. Total number of samples were 53. A distinct
collection up to 8 species from the 24 genera of Pezizales were chosen. Highest number of
samples belongs to the following genea: Sarcoscypha (8), Peziza (6) and Morchella (6).
The measurement results of the spore width and length by using flow cytometer and light
microscope were rather similar. The linear measures of the same spore’ sample in the light
microscope were 30% higher than in the flow cytometer, however, the difference after the
corrections was only 5% (i.e. the mean of the whole measured samples).
The research shows that by studing different problems of systematics the light microscope can
be replaced by flow cytometer, in particular to examine sister and complex species using
equivalent diameter. Also to examine structures of species and fungal population growing in
various geographic locations. Further studies would be needed to research how
ornamentation and asymmetry of fungal spores affect flow cytometer measurements. The
usage of flow cytometer is relevant when measuring concentration of airborne spores that
cause allergic reactions
Application of flow cytometry to measure genome and spore sizes of selected Basidiomycota groups
Get PDFVoolutsütomeetriat 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
Antagonistic effect of selected endophytic fungi against Armillaria spp. in vitro
No full textKülmaseene (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
Nuclear DNA content, life cycle and ploidy in two Neottiella species (Pezizales, Ascomycota)
No full textGenome size, life cycle, and ploidy were determined for Neottiella vivida (Nyl.) Dennis and N. rutilans (Fr.) Dennis. The relative DNA content was measured from fruit-bodies using cytofluorometry; genome size was obtained by comparison with two standards: conidia of Trichophea hemisphaerioides (23.3 Mb) and a sporeprint from Pleurotus ostreatus (25 Mb). Neottiella vivida and N. rutilans were found to have genomes of approximately 750 Mb and 530 Mb, respectively. The ploidy level of N. rutilans is 50, that of N. vivida was calculated to be 70. In N. vivida, meiotic division occurs in the ascus apex where giant mitochondria with a DNA content of 60 Mb, 54 Mb, and 30 Mb were found.
The two species are morphologically very similar and can be distinguished only by their ascospore ornamentation, which is reticulated in N. rutilans and waited in N. vivida. Due to endoreduplication in the uninucleate ascospores of N. vivida, the value of their nuclear DNA content is 2C. In N. rutilans, endoreduplication is not arrested at the 2C value but may proceed at a different rate in spores. Thus N. rutilans reveals heterogeneity in ploidy levels of sporal nuclei. In N. vivida and N. rutilans, differences in spore ornamentation may result from different patterns of gene expression regulated by the ploidy-dependent gene
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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