Project Number: 2072-22000-044-026-G
Project Type: Grant
Start Date: Sep 1, 2021
End Date: Oct 31, 2023
1. Survey of yeast species associated with PNW SWD infesting blueberry; 2. Assess feeding preference of SWD to Hanseniaspora uvarum isolated from blueberry, raspberry, and blackberry compared with wild yeast isolates from blueberry and non-small fruit hosts (NSF); 3. Phagostimulant potential of yeast isolates when combined with insecticides; 4. Fungicide resistance of yeasts isolated from blueberry tissues.
Objective 1. Adult SWD and blueberry plant leaves, flowers, and fruit will be collected from two fields in Whatcom County, WA and one in Pierce County, WA, will be collected 2X monthly throughout the growing season (May - September), with a single collection from Benton County, WA, mid-season. Flies will be vacuum collected, euthanized, surface disinfested, and alimentary canals aseptically dissected and streaked onto chloramphenicol amended Wallerstein Labs nutrient agar. Pure colony cultures of yeast-like morphotypes will be streaked for isolation on potato dextrose agar (PDA). Blueberry leaves, inflorescence, fruit, and other NSF field border plants will be sampled for yeasts, simultaneously with SWD collections. Plant tissues will be macerated in phosphate buffered saline (PBS) and yeast colonies will be isolated and put into pure culture as described above. Yeasts from NSF hosts, outside SWD’s ecological niche, will be evaluated for SWD attraction. Yeast species will be characterized morphologically as described in Kurtzman et al. (2011). Yeast DNA will be extracted for molecular identification by amplifying and sequencing the ITS and D1-D3 LSU regions and compared to GenBank sequences. Objective 2: SWD larvae reared at room temperature and 16:8 hour day/ night photoperiod, on a standard cornmeal/yeast diet (Bellutti et al. 2018, Hamby et al. 2012) with single H. uvarum isolates, and three dominant wild yeasts from the survey, will be selected for oviposition, feeding preference, and larval development. Objective 3: Surface disinfested blueberry shoots with fruit, will be atomized with PBS only, one insecticide, one yeast, or one of nine insecticide + yeast combinations until run-off, air-dried, then placed in a chamber. Even-aged mixed-sex, adult SWD will be introduced into the chambers and mortality evaluated after 24 hours. Eggs per fruit will be counted. Insecticide treatments include spinosad (Entrust), zeta-cypermethrin (Mustang Maxx), and sabadilla alkaloids (Veratran D). HOOK SWD and Agragene will be included for comparison. Insecticides will be applied at the recommended field rate and combined with yeast in a 1:1 v/v solution. The trial will be performed twice, each with four replications. Objective 4: Yeast isolate collections will be evaluated for resistance to fungicides commonly used to manage Botrytis cinerea in PNW fields. Technical grade boscalid (Pristine), iprodione (Rovral), cyprodinil or fludioxinil (Switch), or fluopyram (Luna Tranquility) will be applied to PDA plates amended with salicylhydroxamic acid (SHAM) using a spiral plater and air- dried prior to spreading the yeast suspension onto the plates for colony counts.