28 research outputs found
Virus bronzavosti paradajza – jedan od najdestruktivnijih biljnih virusa
Tomato spotted wilt virus (TSWV) has one of the largest host ranges among plant viruses
and is widespread in all climates. TSWV is responsible for numerous epidemics in many
parts of the world in different crops, mainly vegetables, tobacco and ornamentals. Its highly
polyphagous nature, effectiveness of virus transmission by the thrips as its vectors, rapidity
with which new variants arise, as well as difficulties in controlling the vectors make TSWV
one of the most dangerous plant viruses. The ability of this virus to cause such severe losses
on a broad range of crops, as well as its intriguing biological and molecular characteristics
place TSWV amongst the most extensively studied plant viruses in the world at present.
This paper provides a general overview of TSWV, encompassing all the major aspects of its
biology and current knowledge on host range, symptomatology, molecular biology, vector
relationship, control and diagnosis.Smatra se da virus bronzavosti paradajza (Tomato spotted wilt virus, TSWV) ima najširi
krug domaćina i da je jedan od najrasprostranjenijih biljnih virusa. Ovaj virus je odgovoran
za brojne epidemije na različitim usevima u mnogim delovima sveta, a najčešće na povrću,
duvanu i ukrasnim biljkama. Visoko polifagna priroda virusa, efikasnost prenošenja vektorima
– tripsima, brzina kojom se stvaraju nove varijante virusa, kao i teškoće u kontroli vektora,
čine TSWV jednim od najopasnijih biljnih virusa. Zbog ekonomske važnosti na širokom
krugu domaćina, kao i interesantnih bioloških i molekularnih karakteristika, ovo je danas jedan
od najproučavanijih biljnih virusa. Ovaj pregledni rad o virusu bronazavosti paradajza
bliže objašnjava sve aspekte njegove biologije i sadrži savremene podatke o nekim njegovim
osobinama koje se odnose na niz domaćina, simptomatologiju, molekularnu biologiju,
odnos sa vektorom, kontrolu i dijagnozu oboljenja koje prouzrokuje
Effects of n-3 Fatty Acids Supplementation on Plasma Phospholipids Fatty Acid Composition in Patients with Obstructive Jaundice- a Pilot Study
Nutritional and immunological status of patients with obstructive jaundice is usually severely altered, with high mortality rates. The n-3 polyunsaturate fatty acids (PUFA), particularly eicosapentaenoic acid (EPA, 20:5 n-3), posess potent immunomodulatory activities. Thus, our aim was to compare the plasma phospholipid fatty acid (FA) composition of these patients with healthy subjects, as well as before and after 7 days preoperative supplementation with high doses of EPA (0.9 g per day) and docosahexaenoic acid (DHA, 22:6 n-3, 0.6 g per day). We found impaired FA status in obstructive jaundice patients, especially EPA, DHA and PUFA, but significantly increased content of total n-3 FA, 22:5 n-3 FA and particularly EPA, which increased more than 3 fold, after 7 days supplementation. In addition, the n6/n3 ratio significantly decreased from 14.24 to 10.24, demonstrating severely improved plasma phospholipid profile in these patients after the intervention
Učestalost pojave i molekularna karakterizacija virusa mozaika lubenice poreklom iz Srbije
Watermelon mosaic virus (WMV) is widespread in cucurbit crops, most commonly occuring
in temperate and Mediterranean regions. In Serbia WMV has been detected in single and
mixed infections with Zucchini yellow mosaic virus and Cucumber mosaic virus in field-grown
pumpkin and squash crops. Among pumpkin-affecting viruses WMV is the most frequent
one, both by the number of localities and its incidence at each location. During the growing
season of 2009, samples from 583 plants of Cucurbita pepo cvs. Olinka, Belgrade zucchini
and Tosca (Zucchini group), as well as from C. maxima and C. moschata showing symptoms
of virus infection were collected from 12 commercial fields at eight localities and analyzed
by DAS-ELISA using polyclonal antisera specific to six most important cucurbit viruses.
Interestingly, WMV was detected at fewer sites and had lower incidence rate than in two previous
years. In single infections, WMV was found in 11% of tested plants in three fields; in mixed
infections with ZYMV, it was recorded in 9.9% of plants in five fields and with CMV in only 0.2%
in one field. The partial coat protein gene and 3’ non-translated region from two representative
isolates of WMV originating from different localities and host plant species were amplified
by RT-PCR, sequenced, and compared with the sequences available in GenBank database.
The PCR-amplified fragment of predicted size of approximately 1017 bp was obtained. The
sequences of isolates 137-08 (Acc. No. GQ259958) and 159-08 (GU144020) proved to be 94-99%
identical at the nucleotide level with those from other parts of the world. The sequences of
these two isolates differed from each other only at two nucleotide positions, without any amino
acid substitution. Phylogenetic analysis of 57 isolates based on 750 bp sequences of the
coat protein gene showed no correlation between isolates and their geographic origin, and it
also indicated that these isolates fell into three molecular groups of strains. Serbian WMV isolates
belong to group I, the most widespread group of isolates of this virus in the world.Virus mozaika lubenice (Watermelon mosaic virus, WMV) široko je rasprostranjen u usevima
vrežastih kultura, mada se najčešće javlja na Mediteranu i regionima sa umerenom klimom.
U Srbiji, WMV je detektovan u pojedinačnim i mešanim infekcijama sa virusom žutog mozaika
cukinija (Zucchini yellow mosaic virus, ZYMV) i virusom mozaika krastavca (Cucumber mosaic
virus, CMV) u usevima tikava i bundeva gajenim na otvorenom polju. Od virusa tikava, WMV
je često prevalentan virus, kako po broju lokaliteta u kojima je dokazan, tako i po učestalosti
u samom usevu. Iz ukupno 12 komercijalnih useva na osam lokaliteta, 2009. godine sakupljeno
je 583 biljaka Cucurbita pepo cvs. Olinka, Beogradska tikvica i Tosca (cukini forma), kao i C.
maxima i C. moschata, sa simptomima virusnih zaraza, koje su analizirane na prisustvo šest najznačajnijih
virusa tikava primenom DAS-ELISA metode. WMV je detektovan na manjem broju
lokaliteta i u manjoj učestalosti u poređenju sa prethodne dve vegetacije. U pojedinačnim
infekcijama, WMV je detektovan u 11% testiranih biljaka u tri useva, a u mešanim sa ZYMV u
9,9% testiranih biljaka u pet useva i sa CMV u 0,2% testiranih biljaka u jednom usevu. Deo gena
za protein omotača virusa i deo 3’ neprepisujućeg regiona dva odabrana izolata WMV poreklom
iz različitih lokaliteta i biljaka domaćina umnožen je primenom RT-PCR metode, sekvencioniran
i upoređen sa sekvencama dostupnim u GenBank bazi podataka. Dobijen je PCR
produkt očekivane veličine od 1017 bp. Sekvence izolata 137-08 (Acc. No. GQ259958) i 159-08
(GU144020) pokazale su 94-99% nukleotidne identičnosti sa izolatima WMV iz drugih delova
sveta. Sekvence ova dva izolata razlikovala su se jedna od druge samo u dva nukleotidna mesta
što nije imalo za posledicu aminokiselinske supstitucije. Filogenetske analize 57 izolata, na
osnovu 750 bp sekvence gena za protein omotača, nisu ukazale na korelaciju sa geografskim
poreklom izolata, a pokazala su grupisanje izolata u tri molekularne grupe sojeva. Izolati WMV
iz Srbije pripadaju grupi I, grupi koja obuhvata najrasprostranjenije izolate ovog virusa u svetu
Uticaj neravnomerne distribucije virusa bronzavosti paradajza na serološko dokazivanje virusa u paradajzu, paprici i ukrasnim biljkama
Reliable detection of plant pathogens does not only mean the development of sufficiently
sensitive laboratory techniques for their routine testing. Regardless of the sensitivity of applied methods, the proper selection of samples to be tested has crucial influence on
method reliability. Due to uneven distribution of Tomato spotted wilt virus (TSWV) in naturally
infected systemic host plants, the collection and sampling of material for assaying is a
critical moment upon which the reliability of laboratory procedure depends.
The effect of irregular virus distribution on its serological detection was examined in
tomato, pepper and four ornamental species, as its most important host plants in our country.
The reliability of virus detection, depending on its uneven distribution, was assessed
by serological testing of tomato and pepper symptomatic leaves and fruits, and symptomatic
and asymptomatic young and old leaves, as well as flower petals of ornamentals.
Although TSWV was detected using ELISA in the majority of plants included in the experiment,
the tests indicated an uneven distribution and unequal concentrations of TSWV in
different parts of the plants. The virus could not be detected in a certain number of subsamples,
prepared from infected tomato and pepper fruits and older ornamental leaves.
The virus also could not be detected in some ornamentals and tomato plants with intensive
symptoms. Conversely, the virus was detected in three ornamental plants without any
symptoms. Examining the virus distribution in different plant parts indicated that the reliability
of ELISA could be reached not only by sampling younger ornamental leaves, but also
by preparing compound samples with as much leaves as possible, or by testing a greater
number of subsamples of the tested plant. Considering a small possibility of TSWV detection
in tomato and pepper fruits, the infection of these should be established by testing
their leaves. Besides, the data show that negative test results for the known host plants
with characteristic symptoms require tests to be repeated using more sensitive methods
than ELISA.
These results are important as guidelines for growers and professionals who submit
samples, as well as for laboratories performing routine testing.Pouzdana detekcija fitopatogenih organizama ne podrazumeva samo razvijanje dovoljno
osetljivih laboratorijskih tehnika za rutinsko testiranje. Bez obzira koliko primenjene metode
bile osetljive, na njihovu pouzdanost presudno utiče pravilan izbor uzoraka koji će se
testirati. Za virus bronzavosti paradajza (Tomato spotted wilt virus, TSWV), zbog neravnomerne
distribucije u sistemično zaraženim biljkama domaćinima, prikupljanje i priprema uzoraka
za testiranje predstavljaju kritični momenat od koga zavisi pouzdanost laboratorijske
metode.
Sprovedena su ispitivanja uticaja neravnomerne distribucije virusa u paradajzu, paprici
i četiri vrste ukrasnih biljaka, kao najznačajnijim domaćinima ovog virusa u našoj zemlji.
Pouzdanost detekcije virusa, u zavisnosti od njegove neravnomerne distribucije, utvrđivana
je serološkim testiranjem lišća i plodova paradajza i paprike sa simptomima i simptomatičnog
i asimptomatičnog mlađeg i starijeg lišća, kao i kruničnih listića ukrasnih biljaka.
Iako je primenom ELISA metode TSWV dokazan u većini biljaka uključenih u eksperiment,
obavljena testiranja ukazala su na neravnomernu distribuciju i različitu koncentraciju
TSWV u različitim delovima biljaka. Prisustvo virusa nije moglo biti dokazano u određenom
broju poduzoraka pripremljenih od zaraženih biljaka, plodova paradajza i paprike
i starijeg lišća ukrasnih biljaka. Virus nije mogao biti detektovan ni u nekim ukrasnim biljkama
i biljkama paradajza sa izraženim simptomima. Nasuprot tome, TSWV je dokazan u
tri ukrasne biljke bez simptoma. Ispitivanja distribucije virusa u biljci ukazala su da se pouzdanost
ELISA metode može postići uzorkovanjem ne samo mlađeg lišća ukrasnih biljaka,
već pripremom zbirnog uzorka sa što više listova ili ispitivanjem većeg broja poduzoraka
biljke koja se testira. S obzirom na malu mogućnost detekcije TSWV u plodovima paradajza
i paprike, utvrđivanje zaraženosti ovih biljaka treba obaviti testiranjem lišća.
Pored toga, dobijeni rezultati ukazuju da je u slučaju pojave negativnih rezultata testiranjem
uzoraka sa karakterističnim simptomima na poznatim domaćinima, potrebno ponoviti
testiranje osetljivijim metodama od ELISA. Dobijeni rezultati značajni su kao smernica za proizvođače i stručna lica koja dostavljaju uzorke i laboratorije zadužene za rutinska
testiranja
Novi pravci primene otpornosti tikava u kontroli virusnih oboljenja
As there is a growing frequency of viral plant diseases in epidemic proportions, the
possibilities for successful control are constantly being explored. Despite the fact that integral
and simultaneous employment of numerous control measures may contribute to the
decreasing amount of yield losses, especially concerning non-persistently aphid-transmitted
viruses, these measures are often not efficient enough. Research into the basis of resistance
to viral infection and principles of its inheritance, introduction of sources of resistance
in susceptible genotypes, by conventional or genetic manipulations, are very intensive
for cucurbit crops, especially pumpkins. Pumpkin crops are being endangered by a
great number of different viruses, among which the Zucchini yellow mosaic virus, (ZYMV),
Watermelon mosaic virus (WMV) and Cucumber mosaic virus (CMV) are present every year in
Serbia, frequently causing epidemics.
The majority of pumpkin cultivars are not resistant or tolerant to viral infections, but
sources of resistance have been identified in various related species. So far, the identified
sources of resistance to the ZYMV are found in Cucurbita moschata and Citrullus lanatus var.
lanatus genotypes and consist of one or several major dominant genes of resistance. It is a
similar case with WMV, although the sources of dominant major genes are identified in C.
lanatus and C. colocynthis. The sources of resistance to CMV in the form of one dominant
gene have been identified in the genotype C. moschata, although the introduction of this
gene by conventional means proved to be very difficult. Besides the aforementioned, substantial
efforts are being made in developing genotypes with multiple resistance against
several viruses and even other pathogens, as well as genotypes with resistance to the most
significant plant aphid species, through mechanisms of antixenosis or antibiosis.
The other way of obtaining resistant genotypes includes genetic manipulation.
Genetically modified resistant pumpkins have been among the first successfully developed
crops. Genotypes with pathogen derived resistance can already be found in commercially
grown pumpkins in some parts of the world, and they have been developed by introducing
the coat protein gene of one, two or all three viruses which are the most frequent,
ZYMV, WMV and CMV. Yet, this approach to the control of pumpkin viral diseases is related
to possible negative consequences, mostly through the already detected gene transfer
to wild plants and development of resistant transgenic weeds of unpredictable impact
on the environment.
Improved host plant genetic resistance to viral infections or biological vectors, developed
by conventional or genetic engineering methods, represents the most dynamic and
prominent field of research. It is economically and ecologically the most justified approach
to the control of pumpkin and other plant diseases caused by viruses non-persistently
transmitted by aphids.Kako je pojava brojnih viroza gajenih biljaka u epidemijskim razmerama sve češća, mogućnosti
za uspešnu kontrolu stalno se ispituju. Integralna i istovremena primena brojnih
pojedinačnih mera kontrole, naročito u slučaju virusa koji se prenose biljnim vašima na neperzistentan
način, mogu da doprinesu smanjenju šteta, ali često nisu dovoljno efikasne.
Proučavanja osnova otpornosti i načina nasleđivanja, unošenje izvora otpornosti u osetljive
genotipove, konvencionalnim ili genetičkim manipulacijama, vrlo su intenzivna kod vrežastih
kultura, posebno tikve. Tikve ugrožava veliki broj raznorodnih virusa od kojih se u Srbiji
svake godine javljaju virus žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV), virus
mozaika lubenice (Watermelon mosaic virus, WMV) i virus mozaika krastavca (Cucumber mosaic
virus, CMV), a pojedinih godina izazivaju epidemije.
Većina sorti gajenih tikava ne poseduje otpornost ili tolerantnost na virusne zaraze, ali
izvori otpornosti identifikovani su u različitim srodnim vrstama. Do sada identifikovani izvori
otpornosti na ZYMV nalaze se u genotipovima Cucurbita moschata i Citrullus lanatus var.
lanatus i obuhvataju jedan ili nekoliko major gena rezistentnosti koji se nasleđuju dominantno.
Slična situacija je i sa WMV, mada su izvori dominantnih major gena identifikovani u C.
lanatus i C. colocynthis. Izvor otpornosti na CMV u vidu jednog dominantnog gena identifikovan
je u genotipu C. moschata, mada je unošenje ovog gena konvencionalnim putem bilo
veoma teško. Pored toga, veliki napori ulažu se u dobijenje genotipova sa istovremenom
otpornošću na više virusa čak i drugih patogena, kao i genotipova koji ispoljavaju otpornost
na najznačajnije vrste biljnih vaši, kroz mehanizme antiksenoze ili antibioze.
Drugi pravac dobijanja otpornih genotipova podrazumeva genetičke manipulacije. Genetski
modifikovane otporne tikve su među prvima, od svih gajenih biljaka, uspešno razvijene.
Genotipovi sa PD (pathogen derived) otpornošću već se nalaze u komercijalnoj proizvodnji
tikava u nekim delovima sveta i dobijeni su unošenjem gena za CP (coat protein)
jednog, dva ili sva tri u svetu najraširenija virusa, ZYMV, WMW i CMV. Ipak, ovaj prilaz kontroli
virusnih oboljenja tikava vezan je za uočene moguće negativne posledice, pre svega kroz
već registrovan transfer gena u biljke spontane flore i nastanak otpornih transgenih korova
nepredvidljivog ponašanja i značaja u prirodi.
Poboljšana genetička otpornost biljke domaćina bilo na infekciju virusima, bilo na biološke
vektore, dobijena konvencionalnim ili metodama genetičkog inženjeringa predstavlja
najdinamičnije i najperspektivnije polje istraživanja kao ekonomski i ekološki najopravdaniji
pristup kontroli oboljenja tikava i drugih biljaka koje izazivaju virusi koji se neperzistetno
prenose biljnim vašima
Prisustvo i rasprostranjenost virusa uljane tikve i molekularna detekcija virusa žutog mozaika cukinija
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
Prisustvo i molekularna karakterizacija virusa mozaika lucerke u usevu duvana u Srbiji
Three-year investigation of the presence and distribution of tobacco viruses in Serbia
revealed that Alfalfa mosaic virus (AMV) appeared every year with different frequency in
tobacco crops. During 2008, the presence of AMV was detected in most of the tested samples
(58.82%) and it was the second most common compared to all other viruses which
presence was confirmed in Serbia. In 2006 and 2007, AMV was detected in a significantly
lower percentage (2.80% and 13.64%, respectively). This study showed that Alfalfa mosaic
virus was more commonly found in multiple infections with two, three or even four detected
viruses. Single infections were detected only in 2006, in one tobacco field in the locality
of Futog. During this investigation, a rapid and simple protocol was optimized and developed
for molecular detection of AMV in tobacco leaves, using primers CPAMV1/CPAMV2
and commercially available kits for total RNA extraction as well as for RT-PCR (reverse transcription
- polymerase chain reaction). Using RT-PCR and these primers that flank the
AMV coat protein gene, a DNA fragment of 751 bp was amplified, sequenced, and compared
with the sequences available in GenBank database. The sequence of isolate 196-08
(GenBank Acc. No. FJ527749) proved to be identical at the nucleotide level of 99 to 93% with
those from other parts of the world. Phylogenetic analysis of 27 isolates based on 528 bp
sequences of the coat protein gene did not show correlation of the isolates with their geographic
origin or plant host and showed that these isolates fall into four molecular groups
of strains. Serbian AMV isolate from tobacco belongs to group IV, the group that includes
most of the isolates selected for phylogenetic analysis.Trogodišnjim proučavanjima pojave i rasprostranjenosti virusa u usevu duvana u Srbiji
utvrđeno je da se virus mozaika lucerke (Alfalfa mosaic virus, AMV) javlja svake godine sa
različitom učestalošću. Prisustvo AMV ustanovljeno je u najvećem broju testiranih uzoraka
(58,82%) tokom 2008. godine kada je bio drugi po zastupljenosti u odnosu na sve ostale
viruse čije je prisustvo utvrđeno u Srbiji. AMV je detektovan u znatno nižem procentu
2006. (2,80%) i 2007. godine (13,64%). Tokom ovih istraživanja virus mozaika lucerke češće
je bio prisutan u mešanim infekcijama sa dva, tri ili čak četiri detektovana virusa. Prisustvo
pojedinačne zaraze utvrđeno je samo 2006. godine u jednom usevu duvana na lokalitetu
Futog. U toku ovih istraživanja razvijen je i optimiziran brz i precizan protokol za molekularnu
detekciju AMV u lišću duvana primenom specifičnih prajmera CPAMV1/CPAMV2
i komercijalnih kitova za ekstrakciju ukupnih RNK i RT-PCR (reverzna transkripcija praćena
lančanom reakcijom polimeraze). Korišćenjem ovih prajmera, koji omogućavaju umnožavanje
gena za protein omotača, umnožen je fragment dužine 751 bp, sekvencioniran i
upoređen sa sekvencama dostupnim u GenBank bazi podataka. Dobijena sekvenca izolata
196-08 (GenBank Acc. No. FJ527749) pokazala je 99-93% nukleotidne identičnosti sa izolatima
AMV iz drugih delova sveta. Filogenetske analize 27 izolata, na osnovu 528 bp sekvence
gena za protein omotača, nisu ukazale na korelaciju sa geografskim poreklom izolata
ili biljkom domaćinom, a pokazale su grupisanje izolata u četiri molekularne grupe sojeva.
Izolat AMV iz duvana iz Srbije pripada grupi IV, koja obuhvata većinu izolata odabranih
za filogenetsku analizu
Identifikacija sojeva virusa crtičastog mozaika krompira na duvanu
Five viruses: Potato Virus Y (PVY), Tomato Spotted Wilt Virus, Cucumber Mosaic Virus,
Tobacco Mosaic Virus and Alfalfa Mosaic Virus, of which PVY was predominant, were detected
by serological testing of tobacco samples collected from many localities in Vojvodina in
2006. Viruses are the most important pathogens in tobacco and PVY causes considerable
economic damages all over the world. A PVY population comprises several different strain
groups, strain subgroups and recombinant strains. Among these, PVYN (necrotic strain) and
PVYO (ordinary strain) cause the greatest yield and quality losses in tobacco. Identification of
a prevalent strain in a PVY population is the basis of proper tobacco genotype selection for
resistance against this significant virus.
Typical symptoms caused by PVY were observed by monitoring tobacco crops in our
country in 2006. The symptoms occurred as changes in the general plant appearance, as
well as necrotic areas on leaves, petiols, stems and flowers. The initial symptoms of veinal
necrosis were expanded throughout the leaf, causing reddish-brown (copper) plant color
and premature death of lower leaves. Plants with these symptoms occurred in all monitored
localities and their frequency was high.
In order to understand various epidemiological aspects of the diseases caused by PVY
and to prevent its occurrence and spreading in tobacco crops, it is necessary to properly
identify this virus in time. Biological, serological and molecular identification of the virus
and its prevalent strain was carried out in order to determine tobacco disease ethiology.
The results obtained suggest that this prevalent strain of PVY has been spreading progressively
in our country in recent years.
Although PVYN is widely spread in tobacco crops in Europe, its destructiveness, disease
intensity and wide distribution in Serbia were established only in the last two years.
PVY necrotic strain was detected mainly in single infections, although it was also present in
mixed infections with other tobacco viruses.Serološkim testiranjem uzoraka duvana prikupljenih u više lokaliteta u Vojvodini 2006.
godine detektovani su Potato Virus Y (PVY), Tomato Spotted Wilt Virus, Cucumber Mosaic Virus,
Tobacco Mosaic Virus i Alfalfa Mosaic Virus, pri čemu je PVY bio dominantan. Virusi su najznačajniji
prouzrokovači oboljenja duvana, a virus crtičastog mozaika krompira (PVY) izaziva
ekonomski značajne štete širom sveta. Populaciju PVY čini više različitih grupa sojeva, podgrupa
u okviru određenog soja i sojeva nastalih rekombinacijom. Od opisanih, PVYN (nekrotični
soj) i PVYO (obični soj) prouzrokuju najveće gubitke u prinosu i kvalitetu duvana.
Poznavanje predominantnog soja u populaciji PVY osnova je pravilne selekcije genotipova
duvana otpornih na ovaj značajan virus.
Pregledom useva duvana u Srbiji tokom 2006. godine, uočeni su tipični simptomi koje
izaziva PVY. Simptomi su se ispoljavali u vidu promena opšteg izgleda biljaka, kao i nekrotičnih
promena na lišću, peteljkama, stablu i cvetu. Početni simptomi nekroze nerava su se širili
i zahvatali celu lisku, usled čega su biljke dobile crvenkastosmeđu (bakarnu) boju, a donje
lišće je prevremeno izumiralo. Biljke sa ovakvim simptomima javljale su se u svim pregledanim
lokalitetima, a učestalost pojave bila je veoma visoka.
Kako bi se razumeli razni epidemiološki aspekti oboljenja koje izaziva PVY i sprečila njegova
pojava i širenje u usevu duvana, neophodna je pravilna i pravovremena identifikacija
virusa. U cilju razjašnjavanja etiologije izvršena je biološka, serološka i molekularna identifikacija
virusa i njegovog prevalentnog soja koji je očigledno u progresivnom širenju poslednjih
godina na duvanu u našoj zemlji.
Iako je PVYN široko rasprostranjen na duvanu u Evropi, destruktivnost, visok intenzitet zaraze
i široka rasprostranjenost ustanovljeni su u Srbiji tek poslednje dve godine. Nekrotični
soj PVY detektovan je uglavnom u pojedinačnim infekcijama, mada je, u manjoj meri, dokazan
i u mešanim infekcijama sa drugim virusima duvana
Karakterizacija virusa mozaika krastavca poreklom iz tikava u Srbiji
Cucumber mosaic virus (CMV) is considered one of the most economically important
plant viruses and has a worldwide distribution and a very wide host range including plants
from family Cucurbitaceae. In Serbia, on cucurbits CMV was detected in single and mixed
infections with Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Viruses,
including CMV, are constantly present in cucurbit crops, but their frequency changes
by year and locality. Surveys and sample collections were conducted in cucurbit crops in
the period from 2008 to 2009 at 15 localities in Vojvodina province, and sample testing was
carried out using the DAS-ELISA method and commercially available antisera for six economically
most important cucurbit viruses. In 2008, a total of 51 samples were collected from
13 cucurbit crops of oilseed pumpkin Olinka variety, squash, and bottle gourd and CMV was
detected in a total of 55% of tested samples with symptoms of viral infection. The most common
infectious type was mixed infection with ZYMV and WMV (35.3%), and then mixed
infection with ZYMV (17.7%) and WMV (2%). A total of 599 symptomatic samples of oilseed
pumpkin Olinka variety, zucchini squash varieties Beogradska and Tosca, squash, and winter
squash were collected in 15 cucurbits crops in 2009. CMV was present in 4.4% of total
collected samples, in single infections in 1.3%, and in mixed with WMV or ZYMV in 1.3%, and
1.8%. Five CMV isolates were obtained by mechanical inoculations of N. glutinosa and one
of them was selected for further biological characterization. Test plants which were described
to be hosts of CMV expressed symptoms characteristic for those caused by CMV after
inoculations by isolate 115-08. CMV specific primers Au1u/Au2d were used to amplify an
850 bp fragment using RT-PCR method. Amplified fragment encodes the entire viral coat
protein (CP) gene and partial 5’ and 3’ UTRs of two selected CMV isolates. Amplified fragments
were sequenced and deposited in the NCBI, where they were assigned accession
numbers, HM065510 (115-08) and HM065509 (151-08). The sequences of CMV isolates from
Serbia shared the highest nucleotide and amino acid identity with isolates from subgroup
IA, from 99.5 to 97.4% and 99.1 to 97.4%, and the lowest identities were with the subgroup II
isolates from 66.9 to 64, 5%, from 75.8 to 74.1%.Virus mozaika krastavca (Cucumber mosaic virus, CMV) je veoma široko rasprostranjen i jedan je
od ekonomski najznačajnijih virusa za poljoprivrednu proizvodnju, sa veoma širokim krugom
domaćina u koji spada i veliki broj gajenih biljaka, među kojima i predstavnici familije
Cucurbitaceae. Epidemiologija ovog virusa je veoma složena, zbog čega je i njegova kontrola
otežana. Na tikvama u Srbiji CMV je detektovan u pojedinačnim i mešanim infekcijama sa virusom
žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV) i virusom mozaika lubenice
(Watermelon mosaic virus, WMV). Virusi, pa tako i CMV, su stalno prisutni u usevima tikava, ali se njihova
učetalost menja po godinama i lokalitetima. Pregledi terena i sakupljanje uzoraka tikava
obavljeni su u periodu 2008-2009. godine na 15 lokaliteta gajenja tikava u Vojvodini, a testiranje
sakupljenih uzoraka izvršeno je primenom DAS-ELISA metode i komercijalno dostupnih antiseruma
za šest ekonomski najznačajnih virusa tikava u svetu. Tokom 2008. godine u 13 useva gajenih
tikava sakupljen je ukupno 51 uzorak uljane tikve sorte Olinka, bundeve i vrga, a CMV je detektovan
u ukupno 55% testiranih uzoraka sa simptomima virusnih zaraza. CMV je najčešće bio
detektovan u mešanoj infekciji sa ZYMV i WMV (35,3%), a zatim u mešanoj infekciji sa ZYMV
(17,7%) i sa WMV (2%). Ukupno 599 simptomatičnih uzoraka uljane tikve sorte Olinka, tikvica sorti
Beogradska i Toska, bundeve i muskatne tikve sakupljeno je u 15 useva tokom 2009. godine. U
sakupljenim uzorcima CMV je bio prisutan u ukupno 4,4% testiranih uzoraka. U pojedinačnim infekcijama
u 1,3%, a u mešanim sa WMV ili ZYMV u 1,3%, odnosno 1,8% testiranih uzoraka.
Mehaničkim inokulacijama N. glutinosa dobijeno je pet izolata CMV od kojih je jedan izabran za
dalju biološku karakterizaciju. Odabrani izolat (115-08) izazvao je na test biljkama koje su opisane
kao domaćini CMV pojavu karakterističnih simptoma. Specifičnim prajmerima CMV Au1u/Au2d
uz upotrebu RT-PCR metode umnožen je deo genoma od oko 850 bp koji kodira ceo protein
omotača virusa i delove 5’ i 3’ neprepisujućih regiona dva odabrana izolata CMV. Amplifikovani
fragmenti su sekvencionirani i prijavljeni u NCBI, gde su im dodeljeni pristupni brojevi, HM065510
(115-08) i HM065509 (151-08). Sekvence CMV izolata iz Srbije dele najveću nukleotidnu i aminokiselinsku
sličnost sa izolatima iz podgrupe IA, od 99,5 do 97,4%, odnosno 99,1 do 97,4%, a najmanju
sa izolatima II podgrupe od 66,9 do 64,5%, odnosno od 75,8 do 74,1%
Prisustvo i rasprostranjenost virusa duvana u Srbiji
Infection with a large number of plant viruses could imperil tobacco yield and quality.
Tobacco is a natural host for more than 20 viruses, among which the most important and
economically harmful are tobacco mosaic virus (TMV), tomato spotted wilt virus (TSWV),
cucumber mosaic virus (CMV), potato virus Y (PVY), alfalfa mosaic virus (AMV), tobacco ringspot
virus (TRSV), tobacco each virus (TEV) and tobacco vein mottling virus (TVMV).
The occurence and distribution of tobacco viruses were investigated for 4 years (2002-
2005). During this period many different tobacco growing localities in Vojvodina and central
Serbia were monitored and samples showing virus symptoms were collected. The collected
samples were tested by DAS ELISA using polyclonal antisera, specific for the detection
of PVY, TSWV, TMV, CMV, AMV and TRSV.
The results obtained for the tobacco virus distribution during these four years of investigation
proved the presence of four economically important viruses in our country, whose
frequencies varied from year to year. In 2002, 2003 and 2004, the most frequent was TSWV
(86.84%; 79% and 49.56%, respectively), while in 2005 PVY was prevalent (56.16%). All viruses
detected in the samples tested were present in single or mixed infections. A corellation
was established between the field symptoms on tobacco and the virus causal agents.
The results obtained showed that TSWV and PVY were the most important tobacco
viruses in our country, so that further research of tobacco virus diseases should be directed
towards their more detailed characterization.Prinos i kvalitet duvana mogu da budu ugroženi usled zaraze velikim brojem fitopatogenih virusa. Duvan je prirodni domaćin preko 20 virusa od kojih su najznačajniji i ekonomski najštetniji virus mozaika duvana (Tobacco mosaic virus, TMV), virus bronzavosti paradajza (Tomato spotted wilt virus, TSWV), virus mozaika krastavca (Cucumber mosaic virus, CMV), virus crtičastog mozaika krompira (Potato virus Y, PVY), virus mozaika lucerke (Alfalfa mosaic virus, AMV), virus prstenaste pegavosti duvana (Tobacco ringspot virus, TRSV), virus graviranosti duvana (Tobacco each virus, TEV) i virus šarenila nerava duvana (Tobacco vein mottling virus, TVMV).
Prisustvo i rasprostranjenost virusnih zaraza duvana su ispitivani tokom četiri godine, od 2002. do 2005. Pregled i sakupljanje uzoraka sa simptomima obavljeno je u više različitih lokaliteta gajenja duvana u Vojvodini i centralnoj Srbiji. Sakupljeni uzorci su testirani DASELISA metodom na prisustvo PVY, TSWV, TMV, CMV, AMV i TRSV.
Dobijeni rezultati ispitivanja ukazuju na prisustvo četiri ekonomski značajna virusa duvana u našoj zemlji i to: TSWV, PVY, TMV i CMV, čija je učestalost po pojedinim godinama i lokalitetima bila različita. Tokom 2002, 2003. i 2004. godine najčešće detektovan virus bio je TSWV (86,84%; 70%, odnosno 49,56%), dok je 2005. godine prevalentan virus bio PVY (56,16%). Svi virusi čije je prisustvo utvrđeno u ispitivanim uzorcima nalazili su se u pojedničnim ili mešanim infekcijama. Bez obzira na izraženu varijabilnost u ispoljenim simptomima virusnih zaraza na duvanu, ustanovljena je zavisnost tipa ispoljenih simptoma od virusa prouzrokovača.
Sprovedena ispitivanja pokazala su da su TSWV i PVY najznačajniji virusi duvana u našoj zemlji, tako da buduća ispitivanja viroza duvana treba da budu usmerena ka detaljnijoj karakterizaciji ovih ekonomski štetnih virusa