Viticulture – Pest and Disease Session

Louis Backmann | Katharina Schmidtmann | Pascal Wegmann-Herr | Andreas Jürgens | Maren Scharfenberger-Schmeer

Assessing Different Botrytis cinerea Strains using Molecular Biological Methods

Louis Backmann,* Katharina Schmidtmann, Pascal Wegmann-Herr, Andreas Jürgens, and Maren Scharfenberger-Schmeer
*Institute for Viticulture and Oenology, Dienstleistungszentrum Ländlicher Raum (DLR) Rheinpfalz, Breitenweg 71, Neustadt, 67435, Germany (louis.backmann@dlr.rlp.de)

The fungal pathogen Botrytis cinerea has a tremendous impact on many crops, including winegrapes. It causes specific off-flavors, brownish color, and poor filtrability in must and wine, and can lead to total loss of harvest. Climate change and development of strains resistant to fungicides make it even more challenging to control the disease. To adapt to those problems, it is important to find and develop new methods to detect Botrytis and differentiate strains. Some of these methods are strain differentiation, classification by simple sequence repeats (SSRs), and early detection of the fungus by qPCR. In this ongoing study, strains from different regions, years, and grape varieties were analyzed using SSR markers and evaluated using either agarose gel electrophoresis or capillary sequencer via PCR. Furthermore, a sensitive qPCR method was refined to achieve early detection of the pathogen. In addition, cross-contamination with other grape pathogens, Penicillium expansum, Trichothecium roseum, and Cladosporium spp., was tested to exclude false quantification of the biomass. The qPCR method was also tested with different B. cinerea strains, to test for potential quantification differences between strains. The results demonstrate promising ways to distinguish between strains using both agarose gel electrophoresis and capillary sequencing, as well as to detect an infection with qPCR before it becomes visible on grapes. Cross contamination could be excluded with the tested pathogens. The different Botrytis strains tested in qPCR had no significant affect on quantification of the biomass. The results can be used to further understand and analyze different B. cinerea strain characteristics, such as laccase activity, regional, or annual effects. The early detection method can be used to better prepare growers for an impending infection so that targeted efforts can be made.

Funding Support: Forschungskreis der Ernährungsindustrie E.V. (FEI)

Ina de Souza Pacheco | Richard Redak | Linda Walling | Peter Atkinson

How to Block the Spread of Pierce’s Disease: Gene-Editing of the Pierce’s Disease Vector, the Glassy-Winged Sharpshooter

Ina de Souza Pacheco, Richard Redak, Linda Walling, and Peter Atkinson*
*University of CA – Riverside, Department of Entomology, Riverside, CA, 92521 (peter.atkinson@ucr.edu)

Pierce’s disease is a serious disease of California grapevines caused by a pathogenic bacterium, Xylella fastidiosa, which is transmitted by a xylem-feeding insect, Homalodisca vitripennis (the glassy-winged sharpshooter, GWSS). Current methods to control this invasive pest are expensive, relying on insecticides, quarantine, and eradication. By blocking the ability of GWSS to transmit X. fastidiosa, we can control the spread of Pierce’s disease in a sustainable manner. Our control strategy is feasible due to the powers of clustered, regularly-interspaced, short palindromic repeats (CRISPR)-based gene-editing technologies. CRISPR-based technologies will allow us to generate GWSS strains that are unable to acquire X. fastidiosa from infected plants or transmit X. fastidiosa to healthy grapevines. We have developed these technologies for GWSS. We have used CRISPR technologies to knock out a gene or to insert a gene into the GWSS genome. As proof of principle, we generated the first genetic mutants of GWSS using two eye pigmentation genes, white and cinnabar. We obtained mutants at high frequency, these mutant strains are robust, and we have maintained the strains in our laboratory for over 15 generations. We confirmed that the white and cinnabar mutations are specific to the target sites, which is critical for genetic control strategies. We have also demonstrated that we can integrate DNA fragments and genes into specific target sites in the white and cinnabar genes at high frequencies. This technology has allowed us to establish a platform for rapid screening of gene regulatory sequences in GWSS. We still need tissue-specific gene regulators for our genetic control strategy. With the gene-editing technology firmly established in GWSS, we are now identifying genes that can be used to block GWWS’ ability to acquire or transmit X. fastidiosa. Our goal is to make a Xylella-proof GWSS and end the threat of Pierces’ disease in California.

Funding Support: Our research was funded by the Pierce’s Disease/GWSS Board, California Department of Agriculture and the Animal and Plant Health Inspection Service of the United States Department of Agriculture.

Fungicide Resistance in Powdery and Downy Mildew in Australian Vineyards

Ismail Ismail,* Lincoln Harper, Steven Van Den Heuvel, Anthony Borneman, Fran Lopez Ruiz, and Mark Sosnowski
*SARDI, SARDI, Plant Research Centre, Gate 2A, Hartley Grove, Urrbrae, South Australia 5064, Australia (Ismail.Ismail@sa.gov.au)

Grapevine powdery mildew (Erysiphe necator) and downy mildew (Plasmopara viticola) are significant diseases in Australia and worldwide. Fungicides are the key to managing these diseases. However, frequent use can lead to fungicide resistance. Samples were collected from five states in Australia from 2017 to 2023 and phenotyping and genotyping were used to investigate resistance status. Phenotyping was conducted for six and four fungicide groups for powdery and downy mildew, respectively. Both pathogens had varying levels of reduced sensitivity to most fungicide groups tested, with resistance confirmed for fungicides from quinone outside inhibitors (QoI, group 11) and proquinazid (group 13) for E. necator and phenylamide (group 4) and QoI for P. viticola. Two mutations, G143A and Y136F, were identified in E. necator populations. There were strong relationships between reduced sensitivity to QoIs and the presence of the G143A mutation in the CYTB gene, but not between reduced sensitivity to demethylation inhibitor (DMI) and Y136F mutation in the CYP51 gene. The mutant H242R/Y, associated with resistance to succinate dehydrogenase inhibitors (SDHI, group 7), was not detected, but reduced sensitivity was recorded for this group. G143A was also detected in P. viticola isolates, with a strong relationship between phenotype and genotype for QoI. Techniques are being improved to increase the monitoring capacity for fungicide resistance using rotorod spore traps and other sample collection methods to detect mutations linked with resistance. High-throughput laboratory and in-field qPCR methods are being developed and validated, with high-throughput sequencing to evaluate their capability to rapidly and cost-effectively identify fungicide resistance mutants.

Funding Support: Wine Australia and CRC-SAAFE

Shijian Zhuang, Shunping Ding | Edgar Godoy Monterroso | Qun Sun | Matthew Fidelibus | Philippe Rolshausen

Field Application of Biofungicides to Control Powdery Mildew and Botrytis Bunch Rot on California Winegrapes

Shijian Zhuang, Shunping Ding,* Edgar Godoy Monterroso, Qun Sun, Matthew Fidelibus, and Philippe Rolshausen
*California Polytechnic State University-San Luis Obispo, 1 Grand Ave, San Luis Obispo, CA, 93407 (sding01@calpoly.edu)

Grapevine powdery mildew caused by Erysiphe necator and Botrytis bunch rot, caused by Botrytis cinerea, are the two of the most important fungal diseases for California grape production. Growers are increasingly interested in using biofungicides because of tightening state regulations and the development of fungicide resistance in fungal populations. We initiated biofungicide trials in 2023 in two CA regions: CA Central Coast (San Luis Obispo), using Chardonnay and Pinot noir, and the San Joaquin Valley (Fresno), using Chardonnay and Carignan. At each location, two blocks were selected per variety. Weather stations were installed in the vineyards before budbreak to monitor the real-time powdery mildew risk index. Fourteen treatments included three biofungicides (Bacillus subtilis, Streptomyces lydicus, and extract of Reynoutria sachalinensis) applied at different intervals (weekly, biweekly, and mildew risk index-based), biofungicides rotating with synthetics, synthetic standard only, and an untreated control. Treatments started at bloom. Disease incidence and severity were rated biweekly, approximately one week prior to veraison, and yield and berry chemistry were measured at harvest. Biofungicide by itself did not show a better efficacy than synthetic standard on disease incidence or severity. However, the rotation of bio-fungicide and synthetic showed similar efficacy against disease severity as synthetic standard alone on both varieties at each location. No significant difference in yield or berry chemistry was found between biofungicide and synthetic treatments. The ongoing project will be repeated in 2024.

Funding Support: CDFA-Specialty Crop Block Grant Program and California State University Agricultural Research Initiatives

3:30 pm – 3:50 pm	Assessing Different Botrytis cinerea Strains using Molecular Biological Methods Louis Backmann, Dienstleistungszentrum Ländlicher Raum (DLR) Rheinpfalz, Germany
3:50 pm – 4:10 pm	How to Block the Spread of Pierce’s Disease: Gene-Editing of the Pierce’s Disease Vector, the Glassy-Winged Sharpshooter Peter Atkinson, University of California, Riverside
4:10 pm – 4:30 pm	Fungicide Resistance in Powdery and Downy Mildew in Australian Vineyards Ismail Ismail, South Australian Research & Development Institute
4:30 pm – 4:50 pm	Field Application of Biofungicides to Control Powdery Mildew and Botrytis Bunch Rot on California Winegrapes Shijian Zhuang, University of California, Riverside

Viticulture – Pest and Disease Session

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Speakers

Louis Backmann

Peter Atkinson

Ismail Ismail

Shijian Zhuang

Moderator

R. Keith Striegler