Location: Horticultural Crops Disease and Pest Management Research Unit
Project Number: 2072-22000-045-019-G
Project Type: Grant
Start Date: Sep 1, 2021
End Date: Oct 31, 2023
Objective:
1. Use microbiome-based studies to understand the diversity of grapevine trunk disease pathogens and how factors such as the age and location of a vineyard affect their distribution.
2. Explore how different pruning wound and soil treatments affect the fungal microbiome on wound and soil samples.
Approach:
Objective 1: A mixture of old and young vines were surveyed in both the Rogue Valley and Willamette Valley with ages ranging from 2 to more than 35 years. Based on size, vineyard plots were divided into a representative number of strata. At least 5 vines were flagged per stratum and symptoms were recorded. The vines that were flagged were every 5th, 10th, or 20th vine in a row starting from row 1, vine 1 in each stratum. The number of vines flagged depended on the row length. Each flagged vine was examined and any symptoms it exhibited were recorded.
Each flagged vine has been sampled in order to identify pathogens. Using an electric drill, three holes were drilled into each flagged vine. One hole was towards the top of the vine, another was where the graft union is, and the last hole was at the bottom of the vine, near soil level. Wood tissue was collected off the drill and put into a labeled tube indicating the stratum number and the position of the hole from which tissue was collected. After collection, samples were stored in the -80C freezer.
The sample tissue will be flash frozen in liquid nitrogen and homogenized using a bead mill. After homogenization, DNA will be extracted from 100 mg of tissue sample. All samples will be stored at -20C until ready to use.
Next-generation sequencing. NGS will be used to identify fungi in the samples. ITS primers will be used to generate amplicons and Illumina indexing will be performed to create indexed amplicon libraries. Amplicon libraries will be pooled and sequenced on an Illumina MiSeq.
USEARCH will be used to identify and filter sequences. Sequences will be clustered using the software Quantitative Insights into Microbial Ecology (QIIME).
Objective 2: To identify what effect different pruning practices have on the fungal microbiome, a vineyard will be divided into rows representing three treatments with four replicates per treatment. The first treatment will be to remove all pruning debris from the row. The second treatment will be to leave the pruning debris in the row and to mow with a flail mower. The third treatment will be to leave the pruning debris in the row, mow with a flail mower, and apply Bio-Tam 2.0 to the soil.
Similarly, to understand the effect of pruning wound treatments on fungal microbiome, three treatments will be tested with four replicates per treatment. The first treatment will be the wound treatment with VitiSeal, the second treatment will be with Bio-Tam 2.0, and the third will be a non-treated control. All wound treatments will be applied within 24 hrs of pruning.
Soil samples will be collected every other week using a soil auger. Soil will be collected from each treatment with two samples being collected in each row. Samples will be stored at 4C until DNA extraction.
Wound tissue will be collected every other week from vines that received an application of Bio-Tam 2.0, received an application of VitiSeal, or did not receive any application. Sterilized pruning shears will be used to cut pieces of the tissue. The tissue pieces will be taken back to the lab where they will be grinded and stored at 4C until DNA extraction.
