Vinova loza (Vitis vinifera L.) jedna je od najznačajnijih višegodišnjih kultura u Hrvatskoj i svijetu. Vinovu lozu inficira više od 86 različitih virusa iz 18 porodica i 35 rodova, od kojih su mnogi okarakterizirani kao uzročnici ekonomski značajnih virusnih bolesti. U Hrvatskoj je 2018. godine prvi puta potvrđena prisutnost G-virusa vinove loze (GVG; rod Vitivirus, porodica Betaflexiviridae) i badnavirusa vinove loze 1 (GBV-1; rod Badnavirus, porodica Calimoviridae). S obzirom na pripadnost navedenih virusa rodovima čiji predstavnici uzrokuju ekonomske štete u vinovoj lozi, ciljevi ovog doktorskog rada bili su: za oba virusa razviti precizne i pouzdane molekularne metode detekcije, utvrditi osjetljivost i efikasnost razvijenih protokola, utvrditi sposobnost detekcije i sezonsku dinamiku virusa u inficiranim biljkama, istražiti njihovu rasprostranjenost u Hrvatskoj, okarakterizirati izolate na molekularnoj razini te utvrditi načine prijenosa i domaćine (vrste roda Vitis ili izvan njega).
U razvoju novih protokola za detekciju metodom lančane reakcije polimerazom (PCR) korištena su dva GVG i četiri GBV-1 izolata utvrđena metodom sekvenciranja visoke protočnosti (eng. high throughput sequencing, HTS), zajedno s 11 otprije poznatih GVG te jednim GBV-1 izolatom iz baze GenBank (NCBI). Osjetljivost novorazvijenih PCR protokola ispitana je serijskim razrjeđenjima izoliranih RNA, DNA i TNA, korištenjem komercijalnih kompleta za izolaciju (RNeasy/DNeasy Plant Mini Kit, Qiagen) i metode glicin-EDTA-natrij (eng. glycin-EDTA-sodium, GES), a efikasnosti metoda PCR u stvarnom vremenu (qPCR) standardnim krivuljama. Razvijeni višestruki RT-qPCR protokol za detekciju GVG i GBV-1 primijenjen je za procjenu sposobnosti detekcije i sezonske dinamike virusa u zaraženim biljkama na temelju TNA izolirane metodom GES. Istim metodama istraživana je rasprostranjenost virusa, testiranjem 4327 uzorka vinove loze iz komercijalnih i kolekcijskih vinograda u kontinentalnoj i priobalnoj Hrvatskoj. Protokoli temeljeni na metodi RT-PCR za GVG i PCR za GBV-1 primijenjeni su za dobivanje produkata od 35, odnosno 50 izolata, koji su sekvencirani Sangerovom metodom te filogenetski obrađeni zajedno s izolatima determiniranim metodom HTS u ovom istraživanju i s otprije poznatim izolatima. Nadalje, testirani su sljedeći tipovi prijenosa: prijenos ličinkama lozine štitaste uši (Planoccocus ficus Sign.) i mehaničkom inokulacijom na sjemenjake vinove loze, zeljaste test biljke i korove; prijenosom sjemenom vinove loze; prijenos cijepljenjem „na zrelo“ na drvenaste indikatore Vitis rupestris, Vitis riparia, Kober 5BB i LN 33 te cijepljenje „zeleno na zrelo“ korištenjem zelenih diferenciranih pupova na drvenaste indikatore i vinovu lozu.
Nove početnice i probe za detekciju virusa razvijene su na osnovi kapsidnog proteina (GVG), odnosno regije genoma reverzne transkriptaze (GBV-1), a razvijena metoda RT-qPCR pokazala je 100 puta veću osjetljivost u usporedbi s metodom RT-PCR za GVG i PCR za GBV-1, uz efikasnost detekcija u rasponu od 97,91 % do 107,28 %. Sposobnost detekcije virusa utvrđena je kroz cijelu sezonu, uz najmanju osjetljivost metode RT-qPCR za GVG u razdoblju kretanja vegetacije (travanj, svibanj) te negativne rezultate u tri termina uzorkovanja u istom razdoblju za GBV-1. Ukupno je GVG detektiran u 10,54 %, a GBV-1 u 13,38 % uzoraka, pri čemu je njihova pojavnost potvrđena samo u autohtonim sortama, a rasprostranjenost u kolekcijskom nasadu i komercijalnim vinogradima priobalne Hrvatske te u kolekcijskim nasadima na kontinentu. Filogenetskim analizama utvrđena je identičnost izolata podrijetlom iz istih vinograda/lokacija, sugerirajući lokalni prijenos virusa, pri čemu je za GVG utvrđena nova divergentnost formiranjem dvije nove grupe. Za oba virusa dokazan je prijenos ličinkama lozine štitaste uši na vinovu lozu u 14,63 % za GVG te 60,98 % za GBV-1. Ni jednim istraživanim načinom prijenosa nisu potvrđeni domaćini među zeljastim test biljkama ili korovima, kao ni sposobnost prijenosa sjemenom. Dokazan je prijenos na indikatore (Vitis sp.) u pokusima cijepljenja „na zrelo“ i „zeleno na zrelo“, pri čemu promjene na drvu nisu zabilježene, dok je na listovima biljaka inficiranima s GVG i GLRaV-3 uočeno uvijanje lista i/ili crvenilo/žućenje u ovisnosti o indikatoru.
|Abstract (english)|| |
Grapevine (Vitis vinifera L.) is one of the most important perennial crops in Croatia and worldwide. In addition to numerous fungal and bacterial pathogens, grapevine can also be infected by various viruses. To date, more than 86 viruses from 18 families and 35 genera have been identified as grapevine-infecting viruses, some of which are considered widespread agents of economically important viral diseases. Such viruses are major challenge for grape production in all viticultural regions of the world, as the diseases directly affect yield by reducing the grape quality and quantity, as well as the lifespan of the vines. In the absence of curative measures, virus control is based on preventive measures such as the production of healthy planting material and control of vectors. Control measures for grapevine viruses are based on their ecological and epidemiological characteristics, the most important of which are the modes of transmission and virus-host interactions. Viral studies on Croatian autochthonous grapevine cultivars already revealed high infection rates with economically important viruses, together with recently discovered ones that have not yet been characterized.
In 2018, grapevine virus G (GVG; genus Vitivirus, family Betaflexiviridae) and grapevine badnavirus 1 (GBV-1; genus Badnavirus, family Calimoviridae) were reported from Croatia for the first time using the high-throughput sequencing (HTS) technology. GVG was detected in the Croatian autochthonous cultivars Babica (VB -108, MF993573), Dobričić (VD -102, MF993574), Ljutun (VLJ-178, MF781081) and Vlaška (VVL-101, MF993575) from the Grapevine virus collection (University of Zagreb Faculty of Agriculture). GBV-1 was detected from the same collection in cv. Ljutun (VLJ-178, NC_055481) and Dobričić (VD-102). While GVG was already reported from New Zealand in 2017, GBV-1 was reported from Croatia for the first time. Both viruses could be of economic importance, as they belong to genera comprising viral pathogens causing economically important viral diseases in grapevine. For this reason, and considering that both viruses were reported from valuable Croatian autochthonous cultivars, the objectives of this research were: the development of accurate, sensitive and robust molecular detection methods, determination of their seasonal dynamics, distribution in Croatia, partial molecular characterization, together with determination of their modes of transmission and host range within and out of the genus Vitis.
To develop primers and probes for accurate and robust detection, already known virus isolates from the GenBank (NCBI) were analysed together with two GVG and four GBV-1 isolates revealed by HTS in this study. Using Primer 3 and Geneious softwares, conserved genomic regions were selected in the coat protein region (CP) for GVG and reverse transcriptase (RT) for GBV-1 for the new primers and probes design and the detection by end-point reverse transcription-polymerase chain reaction (RT-PCR) for GVG, PCR for GBV-1, and reverse-transcription real-time PCR (RT-qPCR). To make detection more cost-effective, a multiplex RT-qPCR assay was developed and two DNA/RNA/TNA isolation methods were tested: column-based methods of RNA/DNA isolation using commercial kits from Qiagen (RNeasy/DNeasy Plant Mini Kit) and extraction of TNA based on the glycine-EDTA-sodium (GES) method. In addition, the sensitivity of both assays (end-point and real-time PCR) was tested together with two extraction methods using 10-fold serial dilutions. RT-qPCR assay had 100-fold higher sensitivity compared with end-point RT-PCR (GVG) or end-point PCR (GBV-1) for both GVG and GBV-1, regardless of which RNA and DNA isolation method was used (column-based or GES). Both detection methods for GBV-1, RT-qPCR (detectability down to a dilution of 1:10,000,000) and end-point PCR (1:10,000), were more sensitive when the column-based method was used for DNA isolation, compared with the GES method (1:100,000 and 1:100, respectively). In contrast, detection of GVG by RT-qPCR (1:100,000) and end-point RT-PCR (1:100) showed the same sensitivity regardless of the isolation method used. Analyses of standard curves obtained by RT-qPCR for GVG and GBV-1 showed better reaction efficiency based on DNA
and RNA isolated using Qiagen’s kits (103.85% and 97.91%, respectively) compared to TNA isolated using the GES method (106.87% and 107.28%, respectively).
After verifying the efficiency of molecular methods, the detectability and seasonal dynamics of GVG and GBV-1 were studied in infected plants during the 2019 growing and dormant season, using different plant tissues (dormant season – cortical scrapings, young shoots at the beginning of vegetation, petioles of old leaves during vegetation). Using the GES method for isolation of TNA, both viruses were efficiently detected throughout dormancy and during most of the growing season by multiplex RT-qPCR. However, negative results have been found for GBV-1 early in the season (from shoots in April and from petioles in May). The statistically highest sensitivity (low Cq values) of the RT-qPCR for GVG was determined on petioles collected from June to September and the lowest sensitivity (high Cq values) from April to June. On the other hand, differencess beetween detection abillity for GBV-1 during dormancy and the growing season were not significant.
The distribution study for both viruses in Croatia was performed using the GES extraction method for TNA isolation, followed by multipelx RT-qPCR. During the summer of 2020 and 2021, a total 4,327 samples were collected from grapevines originating from 93 different commercial vineyards and five collection plantations. Field screening revealed an overall infection rate of 10.54% for GVG and of 13.38% for GBV-1. Both viruses were detected along the Croatian coastal winegrowing region in commercial vineyards and collection plantation, but only in autochthonous cultivars, with determined infection rates at specific sites ranging from 2 % to 100 % for GVG and from 1.9 % to 96 % for GBV-1. In the continental winegrowing region, GVG and GBV-1 were detected only in autochthonous cultivars from two collection plantations in Zagreb (University of Zagreb Faculty of Agriculture), but not in commercial vineyards.
The 35 end-point RT-PCR products for GVG and 50 PCR products for GBV-1 were selected by their origin, Sanger sequenced in both directions and phylogenetically processed using MEGA11 software. In addition to the previously mentioned isolates, two GVG isolates and four GBV-1 isolates determined by HTS were used for nucleotide and amino-acid comparison in selected genomic regions (CP for GVG and RT for GBV-1), along with isolates already available in GenBank (NCBI). The identities at the nucleotide (nt) and amino acid (aa) levels for newly discovered GVG isolates in the CP genome region ranged from 89.01-100 % and 96.79-100 %, respectively, whereas for GBV-1 isolates in the RT genome region ranged from 94.13-100 % and 92.74-100 %, respectively. Isolates originating from the same sites/vineyards showed mutual similarity or identity in the CP and RT regions of the genome compared to isolates from other sites, suggesting possible on-site transmission by insect vectors. Phylogenetic analysis of all known GVG isolates (from Croatia, New Zealand, and the United States) resulted in a division into five distinct groups, with two new groups formed consisting of isolates identified in this study revealing previously unknown genetic divergence of GVG in the CP region of the genome.
In transmission experiments using first and second instars of vine mealybug (Planococcus ficus Sign.), 10 instars per plant and 48 hours for acquisition and inoculation access period, only vine-to-vine transmission was successful with rates of 14.63 % for GVG and 60.98 % for GBV-1. In contrast, using the same approach, both viruses were not transmitted to herbaceous test plants (Chenopodium murale L. and Nicotiana benthamiana D.) and weeds commonly found in Croatian vineyards (Amaranthus retroflexus L., Ambrosia artemisifolia L., Chenopodium album L., Galinsoga parviflora Cav., and Abuthilon theophrasti Medik.). Negative results, verified by multipelx RT-qPCR, were also obtained for grapevine seed transmission and mechanical transmission using three different inoculation buffers (phosphate, nicotine and phosphate-nicotine-cisteine).
In addition to vector transmission, transference by dormant grafting was confirmed for both viruses, since they were successfully transmitted to the following rootstocks/indicator plants: Vitis rupestris, Vitis riparia, Kober 5BB (Vitis berlandieri x Vitis riparia) and LN 33 (Couderc 1613 x Vitis berlandieri). Overall infection rates, determined by grafting of dormant canes, were 100 % for GVG and 57.69 % for GBV-1.
In transmission by green grafting using the T-budding technique, GVG and GBV-1 were grafted onto indicators: Kober 5BB, LN 33, 110 Richter ( V. berlandieri × V. rupestris), V. rupestris, V. riparia, and grapevines ‘Chardonnay’ and ‘Cabernet Sauvignon’. GVG was transferred to all previously mentioned plants except V. riparia and V. rupestris, whereas GBV-1 was successfully transferred only to ‘Chardonnay’ and ‘Cabernet Sauvignon’. In the case of GBV-1, no symptoms were observed on leaves or woody cilinder after bark removal. While no symptoms occurred on woody cilinder in the case of GVG, symptoms such as reddening/yellowing and/or leaf rolling were observed on Kober 5BB, LN 33, Chardonnay, and Cabernet Sauvingon. It should be noted that in addition to GVG-infection, presence of GLRaV-3 was also confirmed in all previously mentioned plants, as GVG occurred in co-infection with GLRaV-3 in plants used as a source of buds for grafting.
Considering the overall results, GVG and GBV-1 are widespread viruses in Croatia, especially in the coastal winegroving region. Since they have been transmitted by mealybug vector and grafting, and are not regulated in Croatia, there is a real risk of their further spread in Croatia and beyond. Therefore, future research should focus on demonstrating their impact on grape and wine production, while the data on ecological traits obtained in this study could be useful for developing management strategies to slow their spread in the future.