INTRODUCTION

In spring of 1998, Keith Striegler and I discussed setting up a project at the California State University, Fresno Viticulture and Enology Research Center (VERC) to use DNA markers to check California grapevines for the presence of phytoplasmas, the putative causal agent of grapevine yellows disease (GY). 

Upon review of the resulting CATI grant proposal, we decided to broaden the scope of the project to include the general use of molecular markers for grapevine pathology and improvement. The laboratory we envisioned would serve as a research resource for VERC, other CATI researchers, and the entire university. In addition, we hoped to have the lab service the grower community, performing DNA-based pathogen identification and DNA fingerprinting of grapevine cultivars, for a fee. 

Net receipts from the service portion of the laboratory's activities would be intended to support additional research activities at VERC. Thus, growers could get technical help they needed for a competitive price, while at the same time, supporting VERC.

LABORATORY SET-UP

An initial list of needed equipment and supplies was made, and Anthony Martinez, a Fresno State undergraduate, was hired as a part-time technician to run the laboratory. It took a few months for all of the old equipment and supplies to be removed from the laboratory (the former raisin analysis lab, VERC 123) and for our new equipment to arrive and be set up. The laboratory was then open for business! We have since been fortunate to have graduate students Tracy Affonso and Natasha Sherman join our program, forming a nucleus for DNA-based grapevine projects.

Our laboratory has a thermal cycler for performing the polymerase chain reaction, a sterile hood for microbiological work, equipment for running and analyzing agarose gels, a microentrifuge, a refrigerator and freezers for sample and reagent storage, and other equipment necessary for DNA marker work.

EUTYPA PROJECT

The first project that we have undertaken is in collaboration with Dr. Keith Striegler, Dr. Sanliang Gu, graduate student Christopher Lake, and Dr. Nancy Irelan of the Gallo Biotechnology Program. Keith and his colleagues had an ongoing project at the Vino Farms vineyards in Galt, comparing the effects of different training regimens on horticultural characteristics and the incidence of Eutypa dieback. We have attempted to use a polymerase chain reaction (PCR) technique (using Gallo's proprietary PCR primers provide by Dr. Nancy Irelan) which amplifies specific section of Eutypa DNA many times over, to confirm visual diagnosis of Eutypa symptoms in vines under field conditions and natural inoculum. 

Approximately 180 wood samples were collected in the fall of 1998 from spur positions where canes had shown symptoms the previous spring. Samples were processed according to Gallo's recommended protocol and then used as templates in PCR. Archival template samples are being kept back for future analysis. Initial attempts at amplification failed due to unknown substances present in the samples that were inhibitory to PCR. Upon modification of the procedure and resulting dilution of samples, positive detection of Eutypa has been seen in only seven samples. 

A secondary PCR, using the original PCR product as template, is being considered for more sensitive detection. In addition, in the 1999 season, a more rational approach to sampling will be developed based on Eutypa experts' knowledge of fungal growth and sampling techniques.

DNA FINGERPRINTING PROJECT

In the spring of 1999, graduate student Tracy Affonso commenced a project in the laboratory to develop a new DNA fingerprinting technique for grapevine: ribosomal RNA genes (or rDNA). We believe that the rDNA markers may be useful in conjunction with currently available DNA fingerprinting techniques, and our hope is that we may develop cultivar-specific PCR amplification. We will also use these tools to attempt clonal differentiation in grapevine.

Ribosomal rRNA genes (or ribosomal DNA, or rDNA) are present in all organisms. Plants contain three separate rRNA genes, encoding the 5.8S, 18S, and 26S rRNAs, respectively. These genes are arranged in a cluster such as that shown in Fig. 1 (adapted from Avise, 1994):

Highly conserved coding sequences and the more variable ITS regions, which tend to be shorter in length, make up each repeat unit (Avise, 1994). Detectable variation among genotypes occurs here due to variation in the size of the spacer regions and to variation in restriction sites within coding and spacer regions (Schaal, 1985). 

Plants may have from a few hundred to several thousand copies of this rDNA cluster, arranged in tandem (Grierson and Covey, 1984). The presence of multiple copies of our sequence of interest injects the possibility that heterogeneity within the repeats will make our interpretation of the genetic markers difficult. This is a possibility, but unknown mechanisms of concerted evolution seem to homogenize these and other tandemly repeated sequences in the nuclear genome, and a number of studies in higher plants to study population structure using rDNA variation have been successful (e.g. Appels and Dvorak, 1982; Arnold et al., 1991; Rieseberg et al., 1990).

Tracy has collected samples of 40 cultivars and extracted DNA from 10 so far using the procedure of Wang et al. (1996): Sauvignon Blanc, Cabernet Sauvignon, Pinot Noir, Malbec, Merlot, Zinfandel, Cabernet Franc, Chardonnay, Sangiovese, and Syrah. The DNA is of high yield and quality and should be good template for PCR. Successful amplifications of the rDNA ITS regions have been performed in all samples so far but Pinot Noir. DNA sequencing reactions of hte PCR products were set up using Perkin-Elmer's Big Dye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit (Perkin-Elmer, Foster City, CA) and sent to Iowa State for gel analysis. 

Preliminary sequence analysis of the PCR products indicates (1) that they are indeed rDNA ITS regions (based on sequence similarity to data in GenBank), and (2) that there is sequence variability between grapevine cultivars in the rDNA ITS regions. This variability, or polymorphism, may allow us to design PCR primers that will amplify DNA only from a specific cultivar.

Additional PCR and sequencing reactions will be carried out, and sequences obtained will be compared with each other and with sequences in the GenBank and EMBL databases.

GRAPEVINE YELLOWS PROJECT

Grapevine yellows (GY) is a disease characterized by veinal yellowing, margin curling, berry abortion, and shoot dieback (Fig. 2). The disease causes significant loss of yield in France, Germany, Italy, Israel, Australia, and Virginia. In collaboration with Dr. Tony Wolf of the Virginia Polytechnic Institute, our lab is just now undertaking a project to detect the presence of phytoplasmas (the putative causal agent of GY) in potential insect vectors in Virginia. Insect sweeps are being made in symptomatic vineyards by Dr. Wolf and his people every week, according to a standardized protocol (Purcell and Elkinton, 1980). 

Leafhoppers, which have been proven to serve as GY vectors in Europe, will be classified, frozen, and sent to the Molecular Marker lab for analysis. Using previously established procedures (Vega et al., 1993), we will analyze the insects for the presence of phytoplasmas using PCR. This work is supported by a grant from the Virginia Winegrower's Advisory Board to Dr. Wolf and Dr. Prince.

GROWER SERVICE

A planned major component of the Molecular Marker Lab is to provide pathogen detection/identification services to the grower community. This activity has taken a back seat to setting up research projects during the first year of the lab's operation. Our hope is to set up procedures for this activity to start happening in the spring of 2000.

For information on the Molecular Marker Laboratory at VERC, please contact Dr. Jim Prince at jamespr@csufresno.edu or (559) 278-2559.

ACKNOWLEDGEMENTS

Thanks to the Molecular Marker Lab personnel, Tony Martinez, Tracy Affonso, and Natasha Sherman, for their hard work; to Dr. Nancy Irelan and Dr. Rich DeScenzo of the Gallo Biotechnology Group for providing their proprietary Eutypa PCR primers and technical expertise; to the California Agricultural Technology Institute (CATI), the Virginia Winegrowers' Advisory Board, and the California State University Program for Education and Research in Biotechnology (CSUPERB) for funding; and to the VERC and CATI staff for all of their work.

REFERENCES

Appels, R. and Dvorak, J. 1982. The wheat ribosomal DNA spacer region: its structure and variation in populations and among species. Theor. Appl. Genet. 63:337-348.

Arnold, M.L., Buckner, C.M., and Robinson, J.J. 1991. Pollen-mediated introgression and hybrid speciation in Lousiana irises. Proc. Natl. Acad. Sci. USA 88:1398-1402.

Avise, J.C. 1994. Molecular Markers, Natural History, and Evolution. Chapman & Hall, New York.

Grierson, D. and Covey, S. 1984. Plant Molecular Biology. Blackie (Glasgow and London) (USA: Chapman & Hall).

Purcell, A.H. and Elkinton, J.S. 1980. A comparison of sampling methods for leafhopper vectors of X-disease in California cherry orchards. J. Econ. Etnomol. 73:854-860.

Rieseberg, L.H., Carter, R., and Zona, S. 1990. Molecular tests of the hypothesized hybrid origin of two diploid Helianthus species (Asteraceae). Evolution 44:1498-1511.

Vega, F.E., Davis, R.E., Barbosa, P., Dally, E.L., Purcell, A.H., and Lee, I.-M. 1993. Detection of a plant pathogen in a nonvector insect species by the polymerase chain reaction. Phytopathology 83:621-624.

Wang, X.-D., Wang, Z.-P., and Zou, Y.-P. 1996. An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel. Plant Mol. Biol. Reporter 14:369-373.

 

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CALIFORNIA AGRICULTURAL TECHNOLOGY INSTITUTE - CATI
College of Agricultural Sciences and Technology
California State University, Fresno