Jerry K. Uyemoto

A Comparison Between Serological and Biological Assays in Detecting Grapevine Leafroll Associated Viruses

The efficacy of the serological procedure enzyme-linked immunosorbent assay (ELISA) for detecting grapevine leafroll associated viruses (GLRaV types -1, -2, -3, and -4) was compared with indexing on Vitis vinifera L. cv. Cabernet Franc. Results of the biological assays confirmed the infectious nature of all grapevine sources testing positive by ELISA for GLRaV-1 (9 sources), GLRaV-2 (14 sources), and GLRaV-4 (14 sources), and the noninfectious nature of ELISA-negative grapevines (75 sources). However, among 57 sources testing positive by ELISA for GLRaV-3, or 24 sources with multiple infections, 8 and 1 sources, respectively, were negative by Cabernet Franc assays. Serological assays were repeated on all graft-inoculated indicators and only symptomatic ones reacted positively. Also, the 8 original GLRaV-3 sources that had tested positive by ELISA and negative by bioassay were found positive using immuno-capture/reverse transcription-polymerase chain reaction (IC/RT-PCR). The single multiple-infected source was not available for retesting. The distribution of GLRaV in infected grapevines was tested by assaying 20 to 40 samples per source of 36 plants infected with GLRaV-1, -2, -3, or -4. The incidence of GLRaV-positive canes as determined by ELISA ranged from 0 to 100%, suggesting that GLRaV can be unevenly distributed in chronically infected grapevines.

A putative new ampelovirus associated with grapevine leafroll disease

A putative new ampelovirus was detected in Vitis vinifera cv. Carnelian showing mild leafroll symptoms and molecularly characterized. The complete genome consisted of 13,625 nt and had a structure similar to that of members of subgroup I in the genus Ampelovirus (fam. Closteroviridae). In-depth analyses showed that the virus from cv. Carnelian is the most distinct member of the “GLRaV-4 lineage” of ampeloviruses, which comprises GLRaV-4, -5, -6, -9, and the recently characterized GLRaV-Pr, and GLRaV-De. This virus appears to be a new member of the family Closteroviridae, for which the provisional name grapevine leafroll-associated Carnelian virus is proposed.

Molecular characterization and detection of plum bark necrosis stem pitting-associated virus

SummaryThe complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5′ and 3′ ends. PBNSPaV represents the simplest genome organization in the genus Ampelovirus, family Closteroviridae. The ORFs 1a and 1b encode, respectively, a large polyprotein with a molecular mass (Mr) of 259.6 kDa containing conserved domains characteristic of a papain-like protease, methyltransferase and helicase (ORF1a) and a 64.1-kDa protein of eight conserved motifs characteristic of viral RNA-dependent RNA polymerase (RdRp) (ORF1b). ORF1b is presumably expressed via a +1 ribosomal frameshift mechanism. ORF2 encodes a small 6.3-kDa hydrophobic protein of unknown function. ORF3 encodes a 57.4-kDa protein, a homologue of the HSP70 family of heat shock proteins. ORF4 encodes a 61.6-kDa protein with unknown function. ORF5 encodes a 35.9-kDa capsid protein (CP). Lastly, ORF6 encodes a 25.2-kDa minor capsid protein (CPm). Phylogenetic analyses performed on sequences of the HSP70h RdRp and CP support classification of the virus in the genus Ampelovirus. A real-time TaqMan® RT-PCR assay and a one-step RT-PCR were developed for PBNSPaV detection and compared using three different sample preparation methods.

Etiology and Host Range of a Closterovirus Associated with Plum Bark Necrosis-Stem Pitting Disease

Diseased plum (Prunus salicina) cv. Black Beaut trees developed stem gumming, severe bark necrosis, and stem pitting symptoms on the woody cylinder of the main trunk and scaffold branches. The sucker shoots of the peach (Prunus persica) cv. Nemaguard understocks exhibited oak-leaf patterns, but lacked the wood or bark markings. Other susceptible hosts included almond (Prunus dulcis), sweet (Prunus avium) and Japanese flowering (Prunus serrulata) cherries, and several plum (Prunus salicina) and prune (Prunus domestica) varieties. A purified preparation containing high molecular size dsRNAs was obtained initially from diseased cherry (P. avium × Prunus pseudocerasus) cv. Colt tissues. Healthy preparations were devoid of similar sized dsRNAs. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays with degenerate oligonucleotide Closterovirus primers, designed from the HSP70 gene, were used to amplify two DNA fragments of 0.67 and 0.56 kbp. The larger cDNA product was cloned, sequenced (AF195501), and compared with other viral sequences. Depending on the number of nucleotides used in the comparisons, identities ranged from 77% for Grapevine leafroll associated virus to 3 to 44% for Little cherry virus-1. Specific primers from the 0.67 kbp cDNA sequence were designed and used in subsequent RT-PCR assays. The associated 0.67 kbp HSP70 amplicon of Plum bark necrosis-stem pitting associated virus was detected in all graft-inoculated Prunus species and varieties except prune (P. domestica var. French Improved).

Biological, molecular, and serological studies of a novel strain of grapevine leafroll-associated virus 2

In California, a novel closterovirus was detected in “Redglobe” grapevine, associated with graft incompatibility and given a trivial name “Grapevine rootstock stem lesion associated virus (GRSLaV).” The biological properties of the putative virus were ascertained when asymptomatic yet infected Redglobe scion buds were graft-inoculated onto test plants of Cabernet Sauvignon propagated on 18 different rootstocks. It proved lethal on test plants growing on rootstocks 1616C, 5BB, 5C, 3309C, and 1103 P, whereas latent infections occurred on the remaining scion-rootstock combinations. In contrast, GLRaV-2 type (type strain) produced only typical leafroll symptoms. In a different experiment, GLRaV-2 type was successfully sap-transmitted to N. benthamiana, whereas sap transmission of GRSLaV was unsuccessful. Double-stranded RNA was extracted from infected Redglobe grapevines, cloned, sequenced, and determined a genome length of 16,527 nucleotides. Computer-assisted analysis of open-reading frames (ORFs) revealed a genome organization typical of monopartite viruses in the genus Closterovirus with nine ORFs (range 71–79% identity) with GLRaV-2 type, the closest similar virus species within the family Closteroviridae. Also the 3′-UTR of GRSLaV consisted of 223 nucleotides with an extended oligo(A) tract similar to that of GLRaV-2 type, Beet yellow stunt virus, and Beet yellows virus. Recombinant GRSLaV coat protein was expressed in E. coli, purified, and immunized a rabbit to produce polyclonal antiserum. Serological data matched the molecular data, whereby exposed plant tissue extracts of grapevines infected by both viruses (GRSLaV and GLRaV-2) reacted positively with homologous and heterologous viral antisera but not with healthy grapevine extracts in ELISA and Western blot tests. Based on the comparative sequence data and shared antigens, GRSLaV is now considered a strain of GLRaV-2 and redesignated as Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG). Primers specific for GLRaV-2RG were developed, which did not amplify GLRaV-2 type strain. When both sets of specific primers were used in assays of different grapevine collections, the incidence of the respective viruses varied considerably, e.g., 1.7 and 13.5%, respectively, for GLRaV-2RG and GLRaV-2 type.

Viruses Involved in Graft Incompatibility and Decline

“Graft incompatibility” develops in young grapevines when latent viruses in scion sources are transmitted to hypersensitive rootstocks by grafting. Reverse incompatibility, when the rootstock is the source of a latent virus, has not been observed in grapevine. After transplanting, affected vines exhibit weak vegetative growth and eventual vine death. Grapevine leafroll-associated virus 2 (GLRaV-2) was initially shown to be associated with graft incompatibility, particularly its genetic variants from the RG and PN groups. These cause a hypersensitive response on rootstocks Kober 5BB, 1103 Paulsen, 5C Teleki, 3309 Couderc, and 1616 Couderc. The genetic determinants of incompatibility in these rootstocks, which are derived from Vitis berlandieri and V. riparia, have not been identified. In addition, coinfections of GLRaV-2 in combination with Grapevine virus B (GVB) or of Grapevine leafroll-associated virus 1 (GLRaV-1) in combination with Grapevine virus A (GVA) are also associated with vine decline on certain rootstocks derived from V. riparia. For diseases “110R necrotic union” and “3309C stem necrosis distortion,” causative agents have not been identified. Syrah decline disease may be associated with grapevine rupestris stem pitting-associated virus (GRSPaV). This disease differs markedly from graft-incompatible diseases in that stem marks appear on Syrah scions, not on rootstocks.