Presence and distribution of oilseed pumpkin viruses and molecular detection of Zucchini yellow mosaic virus

Abstract

Over the past decade, intensive spread of virus infections of oilseed pumpkin has resulted in significant economic losses in pumpkin crop production, which is currently expanding in our country. In 2007 and 2008, a survey for the presence and distribution of oilseed pumpkin viruses was carried out in order to identify viruses responsible for epidemics and incidences of very destructive symptoms on cucurbit leaves and fruits. Monitoring and collecting samples of oil pumpkin, as well as other species such as winter and butternut squash and buffalo and bottle gourd with viral infection symptoms, was conducted in several localities of Vojvodina Province. The collected plant samples were tested by DAS-ELISA using polyclonal antisera specific for the detection of six most economically harmful pumpkin viruses: Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMW), Squash mosaic virus (SqMV), Papaya ringspot virus (PRSV) and Tobacco ringspot virus (TRSV) that are included in A1 quarantine list of harmful organisms in Serbia. Identification of viruses in the collected samples indicated the presence of three viruses, ZYMV, WMV and CMV, in individual and mixed infections. Frequency of the identified viruses varied depending on locality and year of investigations. In 2007, WMV was the most frequent virus (94.2%), while ZYMV was prevalent (98.04%) in 2008. High frequency of ZYMV determined in both years of investigation indicated the need for its rapid and reliable molecular detection. During this investigation, a protocol for ZYMV detection was developed and optimized using specific primers CPfwd/Cprev and commercial kits for total RNA extraction, as well as for RT-PCR. In RT-PCR reaction using these primers, a DNA fragment of approximately 1100 bp, which included coat protein gene, was amplified in the samples of infected pumkin leaves. Although serological methods are still useful for large-scale testing of a great number of samples, this protocol, due to its high sensitivity and specificity, is an important improvement in rapid diagnosis of diseases caused by this virus. In addition, the protocol provides a basis for further characterization of ZYMV isolates originating from Serbia.

Intenzivno širenje virusa infektivnih za uljanu tikvu (Cucurbita pepo), poslednjih deset godina imalo je za posledicu značajne ekonomske gubitke u proizvodnji ove kulture koja se gaji na sve većim površinama u našoj zemlji. Kako bi se identifikovali virusi, odgovorni za epidemijsku pojavu i ispoljavanje veoma destruktivnih simptoma na lišću i plodovima uljane tikve, tokom 2007. i 2008. sprovedeno je ispitivanje njihove pojave i rasprostranjenosti. Pregled i sakupljanje uzoraka uljane tikve, kao i nekih drugih vrsta tikava, kao što su bundeva, muskatna i bizonska tikva i vrg sa simptomima virusnih zaraza, obavljeno je na više različitih lokaliteta gajenja uljane tikve u Vojvodini. Sakupljeni uzorci testirani su DAS-ELISA metodom primenom poliklonalnih antiseruma specifičnih za detekciju u svetu šest ekonomski najznačajnijih virusa tikava: virusa mozaika krastavca (Cucumber mosaic virus, CMV), virusa žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV), virusa mozaika lubenice (Watermelon mosaic virus, WMW), virusa mozaika bundeve (Squash mosaic virus, SqMV), virusa prstenaste pegavosti papaje (Papaya ringspot virus, PRSV) i virusa prstenaste pegavosti duvana (Tobacco ringspot virus, TRSV), koji se nalazi na A1 karantinskoj listi štetnih organizama u Srbiji. Identifikacija virusa u sakupljenim uzorcima ukazala je na prisustvo tri virusa, ZYMV, WMV i CMV, koji su se javili u pojedinačnim ili mešanim infekcijama. Njihova učestalost je po pojedinim godinama i lokalitetima bila različita. Tokom 2007. najčešće je dokazan WMV (94,2%), dok je 2008. godine prevalentan virus bio ZYMV (98,04%). Velika učestalost ZYMV u obe godine ispitivanja ukazala je na potrebu za brzom i pouzdanom molekularnom detekcijom ovog virusa. U toku ovih istraživanja razvijen je i optimizovan protokol za detekciju ZYMV primenom specifičnih prajmera CPfwd/CPrev i komercijalnih kitova za ekstrakciju ukupne RNA i RT-PCR. Korišćenjem ovih prajmera, kojim se amplifikuje deo genoma ZYMV kojim je obuhvaćen i gen za proteinski omotač, umnožen je DNK fragment dužine oko 1100 bp iz lišća zaraženih biljaka. Mada serološke metode i dalje imaju veliku prednost u primeni za masovna testiranja velikog broja uzoraka, razvijeni protokol molekularne detekcije, zbog visoke osetljivosti i specifičnosti predstavlja značajno poboljšanje brze dijagnoze oboljenja koja ovaj virus izaziva. Osim toga, ovaj protokol pruža osnovu za dalju karakterizaciju ZYMV izolata poreklom iz Srbije.