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Infektivnost virusa zelene lisavosti in mozaika kumar ter virusa blage lisavosti paprike v vodnih vzorcih vtoka v čistilno napravo Domžale - Kamnik
ID Bačnik, Katarina (Author), ID Dermastia, Marina (Mentor) More about this mentor... This link opens in a new window, ID Ravnikar, Maja (Comentor)

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MD5: C19A28830C2C263B92C47FFE92E85767
PID: 20.500.12556/rul/cc5f55ed-4ed9-4e12-8154-25064138a6b7

Abstract
Analizirali smo vzorec vtoka v čistilno napravo Domžale - Kamnik, da bi ugotovili, če so v vzorcu prisotni patogeni rastlinski virusi. Osredotočili smo se na prisotnost virusa zelene lisavosti in mozaika kumar (Cucumber green mottle mosaic virus; CGMMV) in virusa blage lisavosti paprike (Pepper mild mottle virus; PMMoV). Glavni namen raziskave je bil ugotoviti, ali so virusi CGMMV in PMMoV iz vzorca vtoka, ki smo jih skoncentrirali z uporabo monolitnih kromatografskih nosilcev CIM, infektivni. Prisotnost virusov smo dokazovali z metodo reverzne transkripcije in verižne reakcije s polimerazo v realnem času in ugotovili, da se je njihova koncentracija po koncentriranju osnovnega vzorca povečala za dva velikostna razreda. Infektivnost virusov smo preverjali z mehansko inokulacijo testnih rastlin. Kljub temu, da smo z molekularnimi metodami po koncentriranju dokazali visoke koncentracije virusne RNA in z elektronsko mikroskopijo pokazali nepoškodovane virusne delce, se v razmerah, katerim so bili virusi predhodno izpostavljeni, ni ohranila zadostna koncentracija nepoškodovanih virusov, ki bi lahko uspešno okužili testne rastline. Z rezultati dodatnih kontrol okuževanja smo izključili prisotnost NaCl v vzorcu po koncentriranju z gradientno elucijo kot razlog za odsotnost infektivnosti. Prav tako lahko kot razlog za odsotnost infektivnosti izključimo prisotnost snovi, ki se koncentrirajo skupaj z virusi, saj so dodani infektivni virusi ohranili sposobnost okužbe testnih rastlin. Na celovitost in infektivnost virusov v vzorcu vtoka v čistilno napravo bi lahko vplivale številne snovi, katerim so bili virusi izpostavljeni še pred vzorčenjem in koncentriranjem. V nadaljnjih raziskavah predlagamo uporabo predkolone v postopku koncentriranja virusov in takojšnjo inokulacijo testnih rastlin brez vmesnega zamrzovanja elucijskih frakcij.

Language:Slovenian
Keywords:rastlinski virusi, tobamovirusi, virus zelene lisavosti in mozaika kumar, virus blage lisavosti paprike, infektivnost rastlinskih virusov, odpadne vode, koncentriranje virusov, mehanska inokulacija, molekularne metode, RT-PCR, elektronska mikroskopija
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Publisher:[K. Bačnik]
Year:2017
PID:20.500.12556/RUL-95638 This link opens in a new window
UDC:632.38:578.865:57.083.2
COBISS.SI-ID:4818808 This link opens in a new window
Publication date in RUL:21.09.2017
Views:2827
Downloads:476
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Secondary language

Language:English
Title:Infectivity of c Cucumber green mottle mosaic virus and Pepper mild mottle virus in wastewater treatment plant domžale - Kamnik inflow samples
Abstract:
The presence of pathogenic plant viruses was found in an analysed inflow sample of the wastewater treatment plant Domžale - Kamnik. We focused on detecting the Cucumber green mottle mosaic virus (CGMMV) and the Pepper mild mottle virus (PMMoV). The main purpose of the study was to discover whether the viruses CGMMV and PMMoV from the inflow sample, after having been concentrated with CIM monolithic chromatography, were infective. The CIM monolithic chromatography increased the concentration of viruses for two orders of magnitude. Viruses were detected using the method of reverse transcription real time polymerase chain reaction (RT-qPCR), while infectivity was tested with mechanical inoculation of host plants. Infectivity of the CGMMV and the PMMoV viruses from the inflow sample, concentrated with CIM monolithic chromatography, was not proven. However, we did see intact virus particles in the inflow sample using electron microscopy. Despite the high concentrations detected with molecular methods and intact virus particles seen with electron microscope, the viruses exposed to conditions from the inflow sample did not maintain a sufficient concentration of intact virus particles to infect host plants with mechanical inoculation. Additional controls of inoculation excluded NaCl and the substances from the inflow sample which were concentrated along with the virus particles as reasons for the loss of infectivity. Before the sampling and concentration step, viruses from the inflow sample are already exposed to different substances that can influence their infectivity. For future research we suggest the use of pre-column before concentration step and immediate inoculation of test plants without freezing the elution fractions.

Keywords:plant viruses, tobamoviruses, Cucumber green mottle mosaic virus, Pepper mild mottle virus, infectivity of plant viruses, wastewater, concentration of viruses, mechanical inoculation, molecular methods, RT - PCR, electron microscopy

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