Progress report for GNE24-314
Project Information
The invasion of spotted-wing drosophila (SWD), Drosophila
suzukii (Matsumura), into the United States in 2008 has
inflicted significant damage to soft-skinned fruits such as
blueberries, blackberries, raspberries, strawberries, and
cherries, resulting in annual losses exceeding $500 million.
Presently, Integrated Pest Management (IPM) strategies for SWD
heavily rely on chemical control tactics, which are neither
economically nor environmentally sustainable in the long term.
Thus, it is imperative to develop alternative, more sustainable
IPM tactics for better management of this pest species. One such
underutilized IPM tactic is host-plant resistance. Host-plant
resistance is a key IPM component that leverages the inherent
adaptations of many plants to avoid or tolerate insect or disease
pests and can be harnessed through modern breeding efforts to
develop resistant cultivars. Wild relatives of many domesticated
plants represent an untapped potential source of host-plant
resistance, as studies have shown that they often exhibit greater
resistance to insect pests. However, there has been limited
exploration of wild plants as a potential source of resistance to
SWD. Therefore, the objective of this research is to identify
novel sources of host-plant resistance against SWD by examining
various wild and domesticated blueberry populations. This
endeavor aims to further the development of host-plant resistance
to SWD as a more sustainable IPM tactic. Ultimately, this
research will contribute to the advancement of host-plant
resistance as a viable and eco-friendly strategy for managing
SWD. This proposal aligns with Northeast SARE's Outcome Statement
by focusing on the development of sustainable management
practices for an invasive pest.
Objective 1. Identify novel sources of host-plant resistance to
spotted-wing drosophila (SWD).
I will conduct studies to examine the preference and performance
of SWD on blueberry fruits collected from various wild and
domesticated populations. Based on prior research, I anticipate
that SWD will exhibit a preference for volatiles emitted by wild
blueberry fruits but will show better oviposition and performance
on domesticated fruits. In addition, I expect the strength of
these preference-performance relationships to vary among
different wild and domesticated blueberry populations.
Objective 2. Identify the mechanisms of resistance.
I will conduct studies to evaluate various physical and chemical
fruit traits that may contribute to antibiosis (reduced
performance) and antixenosis (non-preference) resistance across
different wild and domesticated populations. I predict that fruit
size, firmness, sugar and phenolic content, and volatile
emissions will vary among these populations, potentially
correlating with SWD performance and preference.
Objective 3. Provide extension resources to growers.
I will disseminate information on pest management and host-plant
resistance against SWD through presentations at growers'
meetings, articles in newsletters, and the creation of a
factsheet. Additionally, I will establish a timeline for these
extension activities to ensure timely delivery of information to
growers and offer additional support such as training sessions or
expert consultations as needed.
The purpose of this research is to identify novel sources of host-plant resistance against spotted-wing drosophila (SWD), Drosophila suzukii (Matsumura), by examining different wild and domesticated plant populations to further the development of host-plant resistance to this major pest as an Integrated Pest Management (IPM) tactic.
The invasion of SWD into the United States in 2008 has resulted in substantial damage to the country's fruit agriculture (Tait et al., 2021). SWD infestations have been particularly devastating to soft-skinned fruits such as blueberries, blackberries, raspberries, strawberries, and cherries, causing annual losses exceeding $500 million (Bolda et al., 2010). Presently, IPM strategies for SWD heavily rely on chemical control tactics, which are economically and environmentally unsustainable in the long term and can contribute to the development of insecticide resistance (Deans & Hutchinson, 2022). Hence, there is an urgent need to explore alternative, sustainable IPM strategies to effectively manage this pest species. One promising but underutilized tactic is host-plant resistance (Rodriguez-Saona et al., 2019a; Tait et al., 2021).
Host-plant resistance capitalizes on the natural adaptations of plants to deter or withstand insect or disease pests. To implement this strategy, plant breeders identify cultivars or wild relatives of crop plants with desirable resistant traits and cross them with other cultivars to develop plants with both resistance and other desirable traits. Various physical and chemical fruit traits, including size, firmness, sugar content, pH, chemical defense compounds (e.g., phenolics and anthocyanins), nutrient composition, and volatiles, can influence SWD oviposition preference and performance and may thus serve as targets for breeding efforts (Lee et al., 2011; Burrack et al., 2013; Lee et al., 2016; Little et al., 2017; Rodriguez-Saona et al., 2019b; Urbaneja-Bernat et al., 2021; Gullickson et al., 2023). However, many of these traits have been altered during domestication, rendering domesticated fruits more susceptible to pests than their wild counterparts (Macfadyen and Bohan, 2010; Chen et al., 2015a; Chen et al., 2015b). The wild relatives of domesticated plants, which possess a variety of physical and chemical resistance traits, represent an underexplored resource for host-plant resistance against SWD (Agrawal and Fishbein, 2006).
Northern highbush blueberries (Vaccinium corymbosum L., Ericaceae) offer a promising avenue for identifying novel sources of host-plant resistance. Indigenous to the northeastern United States, highbush blueberries were first domesticated in New Jersey in 1911 and have since been selected for traits such as increased fruit size and yield (Ehlenfeldt, 2009; Hancock et al., 2008). The recent domestication history of blueberries allows for a clear comparison of altered traits compared to their wild ancestors, which coexist in regions like the Pinelands National Reserve in New Jersey (McCormick, 1998). While domesticated blueberry varieties are typically tetraploids, wild populations exhibit varying ploidies.
A previous study by Rodriguez-Saona et al. (2019b) found that SWD show a preference for ovipositing in domesticated blueberries and exhibit better performance (measured by adult fly emergence) in domesticated varieties compared to wild ones. This study also revealed alterations in physical and chemical fruit traits due to domestication (Rodriguez-Saona et al., 2019b). However, there have been no studies quantifying the variation in resistance to SWD among populations of wild blueberries. I aim to is to build upon previous research by investigating multiple wild plant populations across different ploidies (diploid and tetraploid) to identify novel sources of resistance against SWD. By exploring natural sources of resistance, I hope to develop sustainable IPM strategies for managing SWD through host-plant resistance. This research aligns with the goals of sustainable agriculture and will contribute to the advancement of host-plant resistance as an effective and environmentally friendly strategy against SWD.
This proposal aligns with Northeast SARE's Outcome Statement by focusing on the development of sustainable management practices for an invasive pest, thereby ensuring the sustainability and economic viability of small fruit growers in the Northeast USA. Through the exploration of host-plant resistance as a sustainable IPM tactic against SWD, this research aims to offer small fruit growers in the region effective and environmentally friendly strategies for managing SWD infestations. By identifying novel sources of resistance among wild blueberry populations and integrating them into cultivation practices, the proposal directly addresses the need for sustainable pest management approaches. Ultimately, the outcomes of this research have the potential to enhance the resilience and profitability of small fruit production in the Northeast USA, aligning with Northeast SARE's objectives of promoting sustainable agriculture practices and supporting the success of regional growers.
Research
- Blueberry genotypes
This experiment will use four blueberry plant types (genotypes): 1) wild tetraploids, 2) wild diploids, 3) domesticated cultivar ‘Duke’, 4) domesticated cultivar 'Bluecrop'. 'Duke' is an early-season cultivar released in 1987 (Draper et al., 1987), while ‘Bluecrop’ is a mid-season cultivar was released in 1952. These cultivars are currently widely planted in New Jersey and in the USA (Hancock, 2001).
- Site selection and sampling method
2.1 Site location
Nine commercial highbush blueberry farms will be selected across New Jersey: four farms located in Atlantic County and five farms located in Burlington County. These farms will be located in the Pinelands region, where most of the blueberry production occur in the state. Each farm will be managed by different landowners and will be under various management practices, including conventional, organic, and U-pick methods. At each farm, the cultivars 'Bluecrop' and 'Duke' will be used as sources of 'domesticated' plants. All blueberry farms selected for this study will have adjacent forested areas where wild blueberry plants are commonly found in the understory. Wild tetraploid and diploid plants discovered in these forested understories adjacent to the farms will thus be utilized as the 'wild' blueberry plants.
2.2 Site sampling
At each site, I will randomly select five bushes for each genotype, totaling 20 bushes per site and 45 bushes per genotype across all farms, with each farm site considered a different population. For each bush, branches will be bagged in the field at fruit set (i.e., green-pink fruit, early June) using porous mesh bags to prevent ambient herbivory by SWD and other pests, as well as exposure to pesticide sprays. At fruit maturation, berries will be harvested from each plant type at each farm. The harvested berries will then be placed in polyethylene ziplock bags inside a cooler and transported back to the laboratory on the same day of collection. These berries will be used for SWD performance and preference assays, as well as fruit trait analyses.
- Experimental design
The experiment will follow a complete randomized block design, with each genotype considered a treatment (fixed effect) and blocked by farm (site). Farm and individual bushes will be treated as random effects.
- SWD Performance/Preference Assays
4.1 Insects
For performance and preference assays, I will employ SWD flies from a lab-reared colony. A SWD colony is currently maintained on standard Drosophila artificial diet (Dalton et al., 2011; Jaramillo et al., 2015, with subtle modifications) at the Rutgers P.E. Marucci Center in Chatsworth, New Jersey. For experiments, I will use sexually matured flies (3–5 days old; Revadi et al., 2015).
4.2 SWD Performance Assays
No-choice laboratory experiments will be conducted to measure the success of SWD offspring in domesticated and wild blueberries. Field-collected berries will be weighed to control for berry size. Equal quantities of fruits (~ 10 g fresh weight) will be added to translucent 2-oz plastic cups lined with moistened filter paper. Ten flies (5 females and 5 males) will be added to each plastic cup, and individual cups will be placed inside rearing cages. Flies within cups will have access to berries for 48 hours. After 48 hours, flies will be removed from the bioassay cups, and number of eggs laid per berry will be counted using a dissecting microscope without damaging the berries; SWD eggs are visible on the surface of fruit skins by observing the oviposition hole and two white breeding filaments protruding out of the egg (Lee et al., 2011). SWD adult survival after 48 h and oviposition will be measured by number of eggs laid per cup.
Egg-infested fruit will then be incubated individually for 14 days post-oviposition to assess adult emergence. Time of adult emergence and number of emerged adults will also be measured per berry. SWD performance will be measured by assessing viable brood and reproductive rate. Viable brood will be calculated (viable brood = number of eggs per cup / number of adults per cup) as well as reproductive rate (reproductive rate = viable brood / berry) to assess offspring success.
All performance assays will be carried out on the same day that fruits will be collected from the field. There will be 5 replicates (1-oz plastic cups) per plant genotype (wild tetraploid, wild diploid, ‘Duke’, and ‘Bluecrop’) per plant farm (9 farms), so N = 180 cups per year. I will repeat these assays for two consecutive years.
4.3 SWD Preference Assays
Choice laboratory experiments will be conducted to measure SWD preference for domesticated versus wild blueberries. Two-choice and 4-choice bioassays will be used to test oviposition preference of SWD females for domesticated versus wild berries. The choice arenas will consist of 10 g berry samples of either wild diploid vs wild tetraploid, wild diploid vs ‘Duke’, wild diploid ‘Bluecrop’, wild tetraploid vs ‘Duke’, wild tetraploid vs ‘Bluecrop’, or ‘Duke’ vs ‘Bluecrop’ (2-choice tests) or 10 g berry samples of all the plant genotypes (4-choice tests). These choice tests will be conducted separately for the berries collected from the different sites to determine differences in SWD preferences for berries across wild and cultivated blueberry populations. Each 10 g berry sample will be placed in a clear 2-oz container, labeled with the sample identifier, and placed in a cage (choice arena). Ten flies (5 females and 5 males) will then be added to each choice arena. Flies will have access to berries for 24 hours. After 24 hours, flies will be removed from the bioassay arenas and the number of eggs laid per berry will be counted using a dissecting microscope.
All preference assays will be carried out on the same day that fruits will be collected from the field. Each choice combinations will be replicated 5 times. I will repeat these assays for two years for a total of 10 replicates per choice combination.
- Berry Traits
5.1 Fruit size
Fruit size (in diameter) will be measured using a dial caliper (Swiss Precision Instruments Inc., Garden Grove, CA, USA). I will use n=10 berries per genotype per site.
5.2 Fruit firmness
Fruit firmness will be measured by the force (in grams) required to puncture the exocarp (FirmTech 2, BioWorks, Wamego, KS, USA). I will use n=10 berries per genotype per site.
5.3 Soluble sugar content (°Brix)
Total soluble solids (°Brix) will be measured from juice extracted from crushed fruit using a digital refractometer (Atago refractometer model PR-32, Atago Co. Ltd., Tokyo, Japan). I will use n=10 berries per genotype per site.
5.4 Acidity
Acidity (pH) will be determined from juice extracted from crushed fruit using a pH meter (model Seven Compact pH /Ion S220; Mettler Toledo, Schwerzenbach, Switzerland) under room temperature (20–25 °C). I will use n=10 berries per genotype per site.
5.5 Total phenolics and anthocyanins
Total phenolics and anthocyanins will be extracted using a method used by Rodriguez-Saona et al. (2019b). Total phenolics will be spectrophotometrically determined with Folin-Ciocalteu reagent according to Slinkard and Singleton (1977)'s method using gallic acid as the standard. Absorbance will be read at 765 nm. Anthocyanin content will be estimated by the pH differential method as mentioned by Giusti and Wrolstad (2001). The absorbance of anthocyanin extracts will be measured in a spectrophotometer at λmax and at 700 nm using the molar extinction coefficient for cyanidin-3-glucoside of 29,600. I will use n=10 berries per genotype per site.
5.6 Total nitrogen and carbon analysis
Mature fruit from each treatment will be freeze-dried and sent to the Penn State University Agricultural Analytical Service Laboratory to be analyzed for total N and C by combustion with an Elementar Vario Max N/C analyzer (Horneck and Miller, 1998). I will use n=5 berries per genotype per site.
5.7 Volatile emissions
Because I am interested in quantifying variation in fruit volatiles among populations of wild and cultivated blueberries that could explain SWD preference and due to constraints in fruit availability, volatile analysis will be done using fruit puree instead of fresh fruit. Harvested blueberry fruits will be washed with distilled water. Ten grams of blueberries will be blended in a glass container with equal weight of distilled water, 20% sodium chloride, and 1% sodium fluoride following a method by Du et al. (2011). Sodium chloride will be employed to reduce possible enzyme activity, and sodium fluoride will be used to reduce microbial growth. The sample will be pureed using a blender for 20 s. Blueberry puree will be placed in vials and stored in freezer at -20°C for later analysis. Headspace volatiles will be collected from each vial and analyzed using gas chromatography flame ionization detection (GC-FID) and gas chromatography mass spectrometry (GC-MS) to quantify and identify the volatile compounds, respectively. I will use n=5 samples (vials) per genotype per site.
- Statistical analyses
Based on results from choice and no-choice tests, I will calculate a host potential index (HPI) following the method of Gullickson et al. (2023) to measure SWD preference and suitability among fruits. Before analysis, all data will be checked for normality (Anderson-Darling test) and equal variances (Levene’s test), and transformed if necessary. Analysis of variance (ANOVA) will be conducted to test for differences among populations of the four genotypes (wild and domesticated blueberries) regarding SWD performance, measured by viable brood (i.e., number of eggs per cup divided by number of adults per cup), and reproductive rate (i.e., viable brood divided by berry). ANOVA will also be used to assess differences in the host potential index among populations of wild and domesticated blueberries. A significant effect of 'site' will indicate differences in SWD preference and performance among populations, while a significant 'genotype-by-site' interaction will demonstrate variation in the effects of population on these preference/performance relationships by genotype.
Similarly, ANOVA will be employed to test for population-level differences between wild and domesticated blueberries in fruit traits, including fruit size, firmness, total soluble solids, pH, anthocyanin and phenolic content, total carbon and nitrogen, and volatile emissions. Additionally, Principal Component Analysis (PCA) will be utilized to visualize population-level differences in various traits among wild and domesticated blueberry fruits.
Education & Outreach Activities and Participation Summary
Participation Summary:
The ideas proposed in this research are innovative and directly relevant to small fruit growers in the Northeast USA, my target audience. Since a significant portion of the research will be conducted at commercial blueberry farms in New Jersey, regular communication with growers will be integral throughout the project. This direct engagement will not only keep growers informed of research progress but also provide an avenue for valuable feedback.
Information on spotted-wing drosophila (SWD) pest management, particularly the potential of host-plant resistance, will be disseminated to growers through various media channels. This includes postings on the Blueberry Bulletin (https://njaes.rutgers.edu/blueberry-bulletin/), a widely subscribed newsletter produced by Rutgers University during the growing season, which covers all aspects of highbush blueberry production and pest management. Additionally, blogs published in the Rutgers Plant & Pest Advisory (https://plant-pest-advisory.rutgers.edu/) will reach a broader audience of small fruit growers.
Furthermore, a factsheet will be developed to provide growers with practical guidance on employing host-plant resistance to manage SWD. Another crucial outreach method involves presenting findings at grower meetings and scientific conferences. I plan to share results from this research at events such as the New Jersey Agricultural Convention & Trade Show, the North American Blueberry Research and Extension Workers conference (expected for 2025; dates yet to be confirmed), and the Entomological Society of America branch and national meetings.
Moreover, I aim to publish 1-2 scientific articles in peer-reviewed journals. These publications will be available to both scientists and farmers and will serve as valuable resources for understanding how host-plant resistance can effectively manage SWD in small fruit crops.