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