Suviodra (Hordeum vulgare L.) seemnetes säilivate fütopatogeensete seente tuvastamine
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Kuupäev
2021
Kättesaadavus
28.08.2021
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Eesti Maaülikool
Abstrakt
Töö eesmärkideks oli leida parim DNA eraldamise metoodika odraseemnetes säilivate patogeensete seente tuvastamiseks ja tõestada polümeraasi ahelreaktsioonil (PCR) põhineva metoodika tõhusust seemnetes säilivate patogeensete seente Pyrenophora teres ja Fusarium liikide tuvastamiseks. Lisaks uuriti seost odraseemnetes säilivate patogeensete seente P. teres ja Fusarium spp. esinemise ja seemnete idanevuse vahel.
Uurimistöö läbiviimiseks koguti 2018.–2020. aastatel Ida-Virumaalt Valaste Agro ja Lüganuse Agro suviodra põldudelt seemneproove. Proovidest määrati seemnete idanevus, hinnati visuaalselt haigussümptomite esinemist ja tuvastati seemnetes P. teres ja Fusarium liikide esinemist erinevaid DNA eraldusmetoodikaid testides.
Seeneliikide tuvastamiseks eraldati DNA kolme erineva metoodika alusel: CTAB, QIAGEN ja SOLIS. Võrreldi metoodikate ajalist ja inimtööjõumahukust, eraldatud DNA kontsentratsiooni ja puhtust lahuses ning fütopatogeensete seeneliikide tuvastamise võimet. Selgus, et DNA kontsentratsioon ja puhtus sõltus olulisel määral DNA eraldusmetoodikast. Metoodikatest oli SOLISel teistest oluliselt kõrgem kontsentratsioon, kuid kehvem DNA lahuse puhtus. Statistiliselt erinevad tulemused DNA metoodikate vahel olid nii universaalse ITS regiooni kui ka spetsiifiliste P. teres praimerite kasutamisel. ITS regiooni praimerite kasutamisel olid kõik QIAGENi ja SOLISe metoodikatega eraldatud DNA-proovid positiivsed ning liigispetsiifiliste P. teres praimerite kasutamisel andis kõige rohkem positiivseid proove SOLISe metoodika. Fusarium liikide tuvastamisel statistilisi erinevusi metoodikate vahel ei tuvastatud, kuid parima tulemuse andis QIAGENi metoodika. Kõige ajamahukam, kuid väikseima tööjõuvajadusega on SOLISe DNA eraldusmetoodika.
Töö käigus analüüsiti veel P. teres ja Fusarium liikide esinemise mõju odraseemnete idanevusele. Statistiliselt parem idanevus esines P. teres´ega nakatunud odraseemnetel. Fusarium liikide esinemine avaldas idanevusele negatiivset mõju.
The aim of the study was to find the best DNA extraction methodology for the detection of phytopathogenic fungi in barley seeds and to prove the effectiveness of the PCR-based methodology for Pyrenophora teres and Fusarium species detection in seeds. Additionally, the relationship between the presence of these phytopathogenic fungi in barley seeds and seed germination was studied. Barley seed samples were collected from the summer barley fields of Valaste Agro and Lüganuse Agro in Ida-Virumaa from 2018 until 2020. Seed germination was determined for the samples. The presence of disease symptoms was visually assessed and the presence of P. teres and Fusarium species in the seeds were detected using PCR methods from DNA samples extracted with three different methods. For phytopathogenic fungi detection DNA was extracted using three different methods: CTAB, QIAGEN and SOLIS. The time and manpower intensity of the DNA extraction methods, the concentration and purity of the DNA in a solution, and the ability to detect phytopathogenic fungal species were compared. It was found that the DNA concentration and purity depended significantly on the DNA extraction method. Of the methods, SOLIS had a significantly higher concentration but poorer DNA purity. There were statistically different results between DNA extraction methods for both the universal ITS region and the specific P. teres primers. When using universal ITS region amplification, QIAGEN and SOLIS gave equally positive samples, and when using P. teres specific primers, SOLIS gave the best results. No statistical differences were found between the DNA extraction methods for the detection of Fusarium species, but QIAGEN gave the best results. The most time-consuming and least labor-intensive is the SOLIS DNA extraction process. In the course of the work, the effect of the presence of P. teres and Fusarium species on the germination of barley seeds was also analyzed. Statistically better germination was observed in barley seeds infected with P. teres. The presence of Fusarium species had a negative effect on seed germination.
The aim of the study was to find the best DNA extraction methodology for the detection of phytopathogenic fungi in barley seeds and to prove the effectiveness of the PCR-based methodology for Pyrenophora teres and Fusarium species detection in seeds. Additionally, the relationship between the presence of these phytopathogenic fungi in barley seeds and seed germination was studied. Barley seed samples were collected from the summer barley fields of Valaste Agro and Lüganuse Agro in Ida-Virumaa from 2018 until 2020. Seed germination was determined for the samples. The presence of disease symptoms was visually assessed and the presence of P. teres and Fusarium species in the seeds were detected using PCR methods from DNA samples extracted with three different methods. For phytopathogenic fungi detection DNA was extracted using three different methods: CTAB, QIAGEN and SOLIS. The time and manpower intensity of the DNA extraction methods, the concentration and purity of the DNA in a solution, and the ability to detect phytopathogenic fungal species were compared. It was found that the DNA concentration and purity depended significantly on the DNA extraction method. Of the methods, SOLIS had a significantly higher concentration but poorer DNA purity. There were statistically different results between DNA extraction methods for both the universal ITS region and the specific P. teres primers. When using universal ITS region amplification, QIAGEN and SOLIS gave equally positive samples, and when using P. teres specific primers, SOLIS gave the best results. No statistical differences were found between the DNA extraction methods for the detection of Fusarium species, but QIAGEN gave the best results. The most time-consuming and least labor-intensive is the SOLIS DNA extraction process. In the course of the work, the effect of the presence of P. teres and Fusarium species on the germination of barley seeds was also analyzed. Statistically better germination was observed in barley seeds infected with P. teres. The presence of Fusarium species had a negative effect on seed germination.
Kirjeldus
Magistritöö
Põllumajandussaaduste tootmise ja turustamise õppekaval
Märksõnad
magistritööd, seenpatogeenid, odrahaigused, molekulaarne identifitseerimine, võrklaiksus, Pyrenophora teres, fusarioos, Fusarium