Development of Low Density PCR Array (LDA) for the Detection of Pathogens in Grapevines.

The goal of this project is to develop a sensitive and reliable molecular method for the detection of viruses in grapevine. This method is called “Low Density PCR Array” (LDA) and is a derivative of real time RT-PCR using TaqMan probe (TaqMan RT-PCR). LDA system uses a fluorescently tagged TaqMan probe and it fluoresces in the presence of target viral RNA in an incremental level following each PCR cycle. The released fluorescent signal then is analyzed by a laser-based thermocycler and evaluated.

In the year 2006-2007 we have optimized the buffer for the 6700 automated Nucleic Acid Workstation for sample preparation and RNA extraction for use in LDA system. The results showed that the 2X ABI lysis buffer (from Applied Biosystems Inc.) worked better than 1X for grapevine tissue and it was comparable to the expensive and time consuming RNeasy column kit produced by Qiagen Inc. The 6700 automated system is quite fast and inexpensive and uses a 96 well plate format and can prepare RNA from 96 samples in approximately 2 hours. In an experiment we tested 29 vines by TaqMan RT-PCR for Grapevine leafroll associated virus (GLRaV) types 2 and 3 by comparing the 1X and 2X lysis buffer and the Qiagen system. In this experiment we found that 15, 12 and 19 plants were tested positive for GLRaV-2 by using Qiagen, 1X and 2X buffer, respectively. For GLRaV-3 the numbers tested positive were 9, 7 and 9 respectively. This comparison revealed that the 6700 automated system produces high quality RNA and was comparable to the RNA extracted by the Qiagen method.

We also designed primers and TaqMan probes for 14 different viruses in grapevine and all have been evaluated for their efficiency in detecting the viruses. These viruses included GLRaV-1 to -7 and -9; GLRaV-2-RG; Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFKV), Rupestris stem pitting associated virus (RSPaV); Grapevine virus A (GVA) and B (GVB). The LDA system developed for these 14 viruses was compared with TaqMan RT-PCR and standard RT-PCR using 29 different grapevine varieties with multiple virus infection from different grape growing regions in the world. The results showed that the LDA method was comparable to TaqMan RT-PCR in detecting viruses and both were superior to RT-PCR. The efficiency of RT-PCR, TaqMan RT-PCR and LDA in detecting viruses in different growing seasons were also compared using five vines each infected with GLRaV-1, -2, -3 and -5 (closteroviruses);GVA and RSPaV (rugose wood complex); GFLV (nepovirus); and GFkV (maculavirus). The results showed that TaqMan RT-PCR and LDA methods were more sensitive than RT-PCR and could consistently detect the viruses in different growing season.

Etiology and Detection of the Cause of Syrah Decline in Syrah grapevine

Syrah decline disease is a well documented problem in California and France. It is characterized by swelling and cracking of the graft union, stem pitting and grooving, and premature leaf reddening. French scientist have been studying this problem since 1999 and have failed to find any correlation with genetic incompatibility, crown gall infection, known pathogens (viruses, fungi, viroids), or environmental conditions. They concluded that the problem had no simple explanation and currently, examining diseased grapevines for a new virus. The potential of a virus causing Syrah decline is reasonable in that trunk stem markings support the hypothesis. In our earlier investigation working with diseased specimens, a new molecularly distinct strain of Rupestris stem pitting associated virus (RSPaV), designated as the Syrah strain, was characterized. However, in follow up assays, we were unable to establish correlations between Syrah decline and RSPaV-SY.

A new genome sequencer, the Genome Sequencer FLX, which is accessible through 454 Life Science Corporation (brandford, CT) offers an opportunity for mass sequencing of cDNA fragments produced from RNA viruses, other microbial organisms or plant related nucleic acids. This system circumvents the bacterial-cloning steps, which is inefficient in retaining clones of viruses in low titers and could sequence thousands of cDNA molecules of 100-300bp in size at a time. In 2007, cDNA samples were prepared from two different Syrah clones, Syrah 6 (showing pitting, wood necrosis and declining symptoms) and Syrah 8 (looks healthy with no visual symptoms), and sent to the 454 sequencing facilities. Total number of fragments (about average of 100-300 nt in size) sequenced by the Genome Sequencer were 169,184 and 182,406 for Syrah 6 and Syrah 8, respectively. Among them, 59,398 and 276 from Syrah 6 and Syrah 8, respectively, belonged to plant viruses and viroids. The viruses that were identified included: RSPaV, Grapevine rupestris vein feathering virus (GRVFV), Grapevine redglobe associated virus (GRGaV), Grapevine leafroll associated virus 9 (GLRaV-9) and a new uncharacterized virus. The viroids included: Hop stunt viroid (HSVd), Australian grapevine viroid AGVd), and Grapevine yellow speckle viroid (GYSVd). Only two viruses that previously have been found by RT-PCR were also found by the 454 sequencing facility in Syrah 8.

Work is in progress to assemble the sequences of all the segments for each virus/viroid and design specific PCR primers for their detection and use the primers to establish a possible correlation between one or multiple of these viruses/viroids with Syrah decline syndrome.

Virus Effects on Fruit and Wine Quality of Cabernet Sauvignon on Six Rootstocks

The objective of this project is to evaluate vine growth, fruit and wine quality of Cabernet Sauvignon when grafted onto six rootstocks with and without viruses. The trial vineyard was established at the UC Oakville Station in Napa Valley, California. Rootstocks include: Couderc 3309, Kober 5BB, Paulsen 1103, Richter 110, Millardet et de Grasset 101-14, and Rupestris St. George. Viruses include: Grapevine leafroll viruses (GLRVs), corky bark associated virus{Grapevine virus B (GVB)}, Grapevine fleck virus (GFkV), and Rupestris stem pitting virus (RSPaV), in single and mixed infections.

This year was the first year in which fruit yield and pruning weight data was collected. Three vines from each replication were measured for a total of 15 vines from each treatment. Parameters measured on fruit were: Fruit yield, cluster number per vine; berry wt (average from a 100?berry sample); fruit maturity indices (Brix, TA, and pH) per?rep; brix will be measured with a refractometer, TA by means of titration with a NaOH standard, and pH by pH meter. The cluster weight and berries per cluster yield components were calculated. Wine was not made because the vines are not fully mature.

Vine size was highly dependent on the rootstock/virus combination. In general, healthy vines were larger than virus-infected vines. At the third leaf stage, summer, 2004, all vines in the healthy treatments in all six rootstocks were rated either medium or large. In contrast, at least 45%of the vines in each virus treatment were rated small on at least one of the six rootstocks. By the 4th leaf in the fall of 2005, healthy vines were still generally larger than leafroll-infected vines; but healthy and RSP-infected vines were similar in size.

On 1103P and St. George, the two rootstocks that had no significant difference in vine growth, there was also no significant difference between healthy and virus-infected treatments in fruit yield. Yield ranged from 1.73 +- 0.29 to 2.03+-0.35 kg/vine for 11093P. On St. George yield ranged from 1.18 to1.52 kg/vine. Yield was significantly reduced in all virus treatments in 110R, although vine size was not reduced as measured by pruning weight. In Kober 5BB, the LR109 treatment yield was extremely low (0.5 kg/vine) but there was no significant difference between other virus treatments and healthy (2.2, 2.1, and 2.1 kg/vine for healthy, LR101, and RSP115, respectively).

As expected, sugar measurement (degrees Brix) was the one parameter in which there was a consistent significant reduction between the two leafroll virus-infected as compared to healthy in all six rootstocks. Treatment LR109 consistently reduced sugars more than LR101. RSP115 had no affect on sugars in 4 rootstocks and slight reduction in St. George and 3309C.

Grapevine Leafroll Associated Virus (GLRaV) types 4 and 6, Molecular Characterization, Biological Study and RT-PCR Detection

Sequencing of the genomic RNA of Grapevine leafroll associated virus (GLRaV) -4 and -6 was continued in 2005. In this effort 4692 and 3832 nucleotides (nt), respectively, for GLRaV-4 and -6 were sequenced. Open reading frames (ORF) identified and the genome organization for each virus included: partial sequence of ORF 1a followed by ORF 1b encoding for a protein recognized as viral RdRp domain. ORF 2 encodes for a small hydrophobic protein followed by ORF 3 encoding for HSP70 homologue protein of 533 aa in length for both viruses. ORF 4 encodes for a putative protein of 539 aa long (P60). ORF 5 encodes for the viral coat protein of estimated molecular mass of 29.5 and 29.2 kDa, and OFR 6, a possible CP minor (CPm) gene, encodes for a putative product of 23.1, and 22.9 kDa, respectively for GLRaV- and -6.

The sequence information revealed that these two viruses are closely related to each other. They have 81%amino acid sequence similarities at the CP region and 78%at the HSP70 region. The phylogenetic analysis using the CP nucleotide sequences of these two viruses compared with other viruses in the family Closteroviridae (the family includes all other known leafroll associated viruses) showed that these two viruses have closer relationship to GLRaV-5 and -9. Specific PCR detection primers have been designed and evaluated.

Development of Polymerase Chain Reaction for Rapid Detection of Grapevine Leafroll Associated Viruses 1 to 5

Specific PCR primers for GLRaVs 1 through 5 were designed, tested and optimized. One step RT-PCR methodology was also developed and the reagents and testing conditions were optimized.
A simple blotting methodology for sample collection in the field was developed. Leaf petioles or young shoots are cut and blotted on specific nylon or nitrocellulose membrane and then these membranes are brought to the laboratory for processing and virus detection. The advantages of this method are: no technical training is required, and if necessary, the samples can be stored for a long time before they are prepared and tested.

In order to develop a reliable RT-PCR detection method, it is very important to identify variations among different isolates (or strains) of each one of these GLRaVs and accordingly design PCR primers that could detect all diverse isolates. In the past year, sequences from the coat protein gene from 10 and 5 different isolates of grapevine leafroll associated virus (GLRaV) -1 and -5, respectively, were compared. The number of nucleotides which were compared from the coat protein gene were 680 and 786 nt for GLRaV-1 and -5 respectively. All GLRaVs-1 isolates used in this study showed 95-99%homology in their compared sequences indicative of a homogeneous population. We had the same observation with GLRaV-5 with again 95-99%homology.

Reliability and sensitivity of RT-PCR for the detection of GLRaVs-1 to -5 was compared with ELISA and with symptom expression of leafroll disease on its biological indicator. From 137 vines tested in this experiment, 26, 16, and 29, respectively tested positive on biological indicator, ELISA, and RT-PCR. The results indicate that the RT-PCR is more sensitive than either indexing on biological indicator host or ELISA. Counting RT-PCR test for GLRaV-5 in this experiment (did not have access to a reliable ELISA reagent for this virus), we found that total of 41 vines were tested positive by PCR which again outnumbers biological index on indicator (27 plants).

PDF: Development of Polymerase Chain Reaction for Rapid Detection of Grapevine Leafroll Associated Viruses 1 to 5

The Role of Single and Mixed Infections of Grapevine Leafroll Associated Virus-2 and Grapevine Virus B in Virus-Induced Rootstock Decline

The first year of this project has been successful in establishing field trials that will enable us to determine susceptibility of rootstock and scion varieties to virus-induced rootstock decline (VIRD); and to correlate virus status with biological effects seen in vineyards. Approximately 2 acres have been planted with 3000 plants that were chip bud inoculated by hand with virus for 11 randomized experiments.

In the first set of trials, 7 rootstocks and two scion varieties were inoculated by chipbudding with 6 virus treatments. Freedom was inoculated as a positive control for the VIRD effect. Nine grape varieties – AXR, St. George, Harmony, 101-14 Mgt, SO4, Kober 5BB and 110R, Cabernet Franc and Chardonnay were inoculated with six virus treatments each. They were planted in a randomized complete block design (RCBD) with 5 blocks and 8 plants/treatment/block for an overall total of 2160 plants. Bud take and baseline data was taken.

In the second set of trials, Freedom and Kober 5BB rootstocks (both susceptible to VIRD) were inoculated with single virus infections and artificial mixes of the single infections to assess the effects of single versus multiple virus infections on growth. They were planted in the field in an RCBD with 5 blocks and 8 plants/treatment/block for a total of 880 plants. Bud take and baseline data was taken.

Extensive PCR testing for 15 grapevine viruses has been done on a selection of virus sources to further identify and characterize naturally-occurring sources of multiple and single virus infections.

PDF: The Role of Single and Mixed Infections of Grapevine Leafroll Associated Virus-2 and Grapevine Virus B in Virus-Induced Rootstock Decline

Development of Polymerase Chain Reaction for Rapid Detection of Grapevine Leafroll Associated Viruses 1 to 5

Specific PCR primers for GLRaVs 1 through 5 were designed, tested and optimized. One step RT-PCR methodology was also developed and the reagents and testing conditions were optimized. Beginning last year we have started testing newly introduced and tissue cultured grape materials using this one step RT-PCR methodology for the detection of these viruses. The information also has been transferred to two different private laboratories in Northern California for their use in testing grower’s material, and another laboratory established by a nursery operation.

In our experience we found that RT-PCR still gives false negative results. We found that sometimes the titer of virus in grapevine tissue is lower than detection threshold, especially on many different rootstocks. However, to improve the sensitivity of RE_{CR for the detection of GLRaVs, we developed nested RT-PCR methodology. We designed nested primers for these viruses and our preliminary test results indicated that this method was 10-100 folds more sensitive that regular RT-PCR. We are in the process of optimizing conditions for this method.

In order to develop a reliable PCR detection method, it is very important to identify variations among different isolates (or strains) of each one of these GLRaVs and accordingly design PCR primers that could detect all diverse isolates. In the past year sequences from the coat protein gene from 15 different isolates of GLRaV-s were compared. It was found that 13 of the tested isolates were very similar in their coat protein sequences, ranging from 99.5%to 100%nucleotide sequence similarites among them. These 13 isolates had 89.2%similarites to the published GLRaV2 coat protein sequence. The remaining two isolates were more divergent and they had 89.4%abd 89.7%similarities to other 13 isolates and 99.5%and 92.5%similarities to the published GLRaV2.

PDF: Development of Polymerase Chain Reaction for Rapid Detection of Grapevine Leafroll Associated Viruses 1 to 5

Grapevine Fleck Virus: Molecular Detection and Investigation of Strain Variability

Specific PCR primers were designed to amplify the coat protein gene of GFKV to use in sequence comparisons of different isolates. Amplification by regular PCR was attempted two times and failed. Finally we designed another pair of primers, internal to the first set and used in nested RT-PCR and successfully amplified the majority of the genome (592 nt out of 672 nt for the full length of the coat protein gene). This nested RT-PCR was tried on 23 different GFKV isolates and it was successful on 17 of them. The sequence comparison among these 17 isolates revealed that they were quite homogeneous and had similarities between 95% to 99.5%.

We have found that nested RT-PCR is more sensitive than regular PCR. Although it requires an additional step to complete, but in our preliminary experiments it was shown to be tens of times more sensitive than regular RT-PCR. Based on its sensitivity and reproducibility, the nested RT-PCR will have a great potential for use in testing grape materials for virus detection.

PDF: Grapevine Fleck Virus: Molecular Detection and Investigation of Strain Variability

Development of Polymerase Chain Reaction for Rapid Detection of Grapevine

Specific PCR primers for GLRaVs 1 through 5 were designed, tested and optimized. One step RT-PCR methodology was also developed and the reagents and testing conditions were optimized. Beginning last year we have started testing newly introduced and tissue cultured grape materials using this one step RT-PCR methodology for the detection of these viruses. The information also has been transferred to two different private laboratories in Northern California for their use in testing grower’s material, and another laboratory established by a nursery operation. In our experience we found that RT-PCR still gives false negative results. We found that sometimes the titer of virus in grapevine tissue is lower than detection threshold, especially on many different rootstocks. However, to improve the sensitivity of RE_{CR for the detection of GLRaVs, we developed nested RT-PCR methodology. We designed nested primers for these viruses and our preliminary test results indicated that this method was 10-100 folds more sensitive that regular RT-PCR. We are in the process of optimizing conditions for this method. In order to develop a reliable PCR detection method, it is very important to identify variations among different isolates (or strains) of each one of these GLRaVs and accordingly design PCR primers that could detect all diverse isolates. In the past year sequences from the coat protein gene from 15 different isolates of GLRaV-s were compared. It was found that 13 of the tested isolates were very similar in their coat protein sequences, ranging from 99.5%to 100%nucleotide sequence similarites among them. These 13 isolates had 89.2% similarities to the published GLRaV2 coat protein sequence. The remaining two isolates were more divergent and they had 89.4%abd 89.7% similarities to other 13 isolates and 99.5%and 92.5%similarities to the published GLRaV2.

Grapevine Fleck Virus: Molecular Detection and Investigation of Strain Variability

Specific PCR primers were designed to amplify the coat protein gene of GFKV to use in sequence comparisons of different isolates. Amplification by regular PCR was attempted two times and failed. Finally we designed another pair of primers, internal to the first set and used in nested RT-PCR and successfully amplified the majority of the genome (592 nt out of 672 nt for the full length of the coat protein gene). This nested RT-PCR was tried on 23 different GFKV isolates and it was successful on 17 of them. The sequence comparison among these 17 isolates revealed that they were quite homogeneous and had similarities between 95% to 99.5%. We have found that nested RT-PCR is more sensitive than regular PCR. Although it requires an additional step to complete, but in our preliminary experiments it was shown to be tens of times more sensitive than regular RT-PCR. Based on its sensitivity and reproducibility, the nested RT-PCR will have a great potential for use in testing grape materials for virus detection.