Understanding Spotted Wing Drosophila’s Role as a Vector for Fruit Rot Fungi in Fall Red Raspberries

Final report for GNE18-178

Project Type: Graduate Student
Funds awarded in 2018: $14,994.00
Projected End Date: 04/30/2021
Grant Recipient: University of Maryland, College Park
Region: Northeast
State: Maryland
Graduate Student:
Faculty Advisor:
Dr. Kelly Hamby
University of Maryland College Park
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Project Information

Summary:

Interactions between plant pathogenic fungi and fruit-feeding insects can play an important role in fruit disease epidemiology. Feeding and egg-laying wounds create opportunities for fungal colonization, and adults can increase disease incidence by passively dispersing fungal propagules. In raspberries, these vectoring interactions may amplify damage from the invasive vinegar fly, spotted-wing drosophila (Drosophila suzukii; SWD). Periods of peak SWD activity overlap with several primary fruit rot pathogens, most notably Botrytis cinerea and Cladosporium cladosporioides, and larvae are known to co-occur with and feed on both Botrytis and Cladosporium at low rates, indicating a possible association that may be exploited for more effective and sustainable pest management. This project lays the groundwork for developing integrated insect and pathogen management programs by generating foundational knowledge about SWD’s fungal interactions.

Field and laboratory-based approaches were utilized to assess potential vectoring relationships between SWD and caneberry (raspberry and blackberry) fruit rot fungi. Specific research aims included conducting laboratory vectoring assays; these assays tested SWD’s ability to acquire and transmit both Botrytis and Cladosporium to sterile media under “worst-case scenario”, no-choice laboratory conditions. Flies carried fungi on both their cuticle and within their digestive tract (indicating feeding on fungi), and nearly 100% of the individuals tested were able to transmit Botrytis and Cladosporium to sterile media for at least 72 hours after their initial exposure to fungi. Field surveys were also conducted to determine whether field populations of adult flies naturally acquire and carry plant pathogenic fungi on their body, a necessary step for vectoring. Over three years, fungi were isolated and molecularly identified from field collected adult SWD. Survey results indicate that SWD associates with a rich fungal community, and flies carry known pre-harvest raspberry pathogens (e.g Cladosporium cladosporioides and Colletotrichum fiorinae) on their body at low rates. Most fungi were detected infrequently. The more commonly isolated genera (Cladosporium and Aspergillus) occurred on less than 50% of the flies sampled, suggesting that SWD do not form tight associations with specific filamentous fungi. Instead, flies may unintentionally pick up fungal spores on their cuticle while resting or feeding on infected substrates. Although passive, this vectoring relationships still has the potential to impact raspberry disease epidemiology.

 Project results were broadly disseminated to various audiences, including presentations to small fruit growers, university extension personnel, and other stakeholders at virtual and in-person extension meetings. Additional presentations to researchers were made at various national and regional scientific conferences throughout the project period. The COVID-19 pandemic restricted access to research facilities and reduced available labor for this project; therefore, we were unable to evaluate how fungal presence and age impacts SWD egg-laying. Overall, the results from this project serve as a proof-of-concept for potential vectoring relationships between SWD and fruit rot fungi. Additional work will be necessary to understand how well these vectoring relationships function under field conditions. Future studies will need to consider various confounding factors that include variable weather conditions, variation in fly behavior under choice and no-choice scenarios, and how competition between fungi may affect the onset of raspberry fruit rot disease. Taking the time to fully characterize these interactions will benefit raspberry production in the long term. If an epidemiological link between SWD and fruit rot fungi exists, we may be able to develop management strategies that synergistically target insect and fungal pests, improving overall sustainability and reducing raspberry production costs.

Project Objectives:

I used a combination of field and laboratory-based approaches to evaluate how interactions between Botrytis and Cladosporium fruit rots and spotted-wing drosophila (Drosophila suzukii; SWD) impacted insect and fruit rot disease dynamics in caneberries (raspberries and blackberries). Specific objectives included:

  1. Evaluating SWD’s ability to acquire and transmit Botrytis or Cladosporium propagules under laboratory conditions. These experiments provided a proof-of-concept for SWD’s vectoring ability and will justify future, field-based experiments that further examine SWD’s impact on fruit rot disease epidemiology.
  2. Surveying field-collected SWD for associations with plant pathogenic fungi, to determine the frequency and mechanism by which adult flies acquire and carry fungal propagules under field conditions
  3. Assessing how Botrytis and Cladosporium fruit rot impact SWD’s oviposition behavior. Understanding these behavioral impacts will help us understand how frequently adult flies encounter and potentially acquire Botrytis or Cladosporium propagules under field conditions.
Introduction:

The purpose of this project was to evaluate how spotted-wing drosophila (Drosophila suzukii; SWD) impacts the disease epidemiology of fruit rot fungi in fall red-raspberries by assessing potential vectoring relationships between SWD and two common fungal pathogens, Botrytis cinerea and Cladosporium cladosporioides. Primocane (fall-bearing) red raspberries are a small, but important component of fruit production in the northeastern United States. Typically planted on diversified fruit farms to extend the raspberry growing season, fall raspberries fill an important gap in the harvest schedule between other summer and fall-fruiting crops. However, they are vulnerable to both insect pests and fungal pathogens.

Raspberry pest management currently primarily focuses on two organisms: SWD, an invasive fruit fly, and Botrytis cinerea (grey mold). However, other fruit rot fungal pathogens may also impact raspberry production. Cladosporium fruit rot, caused by the Cladosporium cladosporioides species complex, is currently considered a minor post-harvest pathogen of ripe or overripe fruit. However, 2016 surveys of Maryland raspberries have reported pre-harvest infection rates as high as 30%, suggesting that Cladosporium may have a more significant economic impact than previously believed. Likewise, anthracnose fruit rot, caused by Colletotrichum fiorinae, has recently been confirmed as a pathogen in Maryland raspberries, with pre-harvest Colletotrichum infection rates as high as 16% reported in unsprayed fields.

Previous research has demonstrated that larval SWD co-occur with and feed on both Botrytis and Cladosporium in raspberries, indicating an association between these pests. However, many aspects of their interactions remain unclear, including the extent to which SWD impacts fungal disease incidence and severity. An epidemiological link between SWD and raspberry fungal pathogens could increase pre-harvest fruit rot incidence and/or severity, consequentially reducing marketable yield. Indeed, interactions with SWD may partially account for the recently observed increase in pre-harvest Cladosporium raspberry infections. Through more precise management of SWD, raspberry producers may be able to minimize crop loss and control fruit rot fungi with fewer fungicides. In addition to increasing profitability (through greater yields and reduced costs for fungicides), this would also increase agricultural sustainability by reducing chemical inputs and delaying the development of fungicide resistance.

Research

Materials and methods:

Objective 1: Evaluate the ability of SWD to acquire and transmit Botrytis or Cladosporium propagules under laboratory conditions.

Using field-isolated fungi, we quantified SWD’s ability to acquire and transmit Botrytis and Cladosporium propagules under “worst-case scenario” no-choice laboratory conditions. 30 male and 30 female SWD were placed directly onto sterile potato dextrose agar (PDA) plates that had previously been inoculated with (1) 200 uL of a Botrytis cinerea spore suspension, (2) 200 uL of a Cladosporium cladosporioides spore suspension, or (3) not inoculated with any fungal pathogens (untreated control). SWD were left on these fungal exposure plates for five hours, during which time they were forced to interact with the fungi. At the end of this exposure period, all flies were removed from the media and evaluated for vectoring ability through the experiments described below in Objectives 1a and 1b. Six replicate assays were conducted for each fungal treatment.

Objective 1A: Quantification of gut and cuticle propagule accumulation

We quantified the density of Botrytis and Cladosporium propagules on adult SWD at four time points: 0, 24, 48, and 72 hours after exposure to the fungal cultures. Including these time points allowed us to quantify the rate at which adult flies accumulate fungal propagules and the length of time that the propagules persisted after the initial exposure. At each time point, four flies (two male and two female SWD) were randomly selected and individually analyzed for the presence and density of fungi located on/in the exterior and interior (e.g. ingested fungi) regions of their body. Individual flies within a time point were treated as subsamples and pooled for the purposes of data analysis.

For the “0-hour” time point, flies were directly removed from the fungal culture plate and transferred into individual sterile microcentrifuge tubes for immediate analysis. The remaining SWD were also removed from the fungal culture plate and transferred onto sterile PDA, where they were held for 24 hours (Figure 1). After 24 hours, we removed an additional four SWD for the “24-hour” post exposure time point and again transferred the remaining flies to a new, sterile PDA (Figure 1). This process was repeated twice more to generate the “48-hour” and “72-hour” time points. Transferring flies to fresh PDA plates at 24-hour intervals helped minimize potential contamination from fungal microbes that could grow on PDA.

Figure 1: Visual summary of methods used to quantify fungal propagule accumulation over time.

At each time point, individual SWD were first submerged in 300 uL of a sterile phosphate buffer solution (PBS) and vortexed for one minute; this dislodged any fungal propagules that had accumulated on the exterior surface of the fly’s body. Each fly was then transferred to a new microcentrifuge tube and surface-sterilized in 95% ethanol for five minutes, followed by two rinses in a sterile buffer solution. This ensured that the exterior fungi did not contaminate isolations from the interior of the fly. Flies were then transferred into a fresh tube of PBS buffer and homogenized using a sterile pestle. To confirm that all surface-dwelling microbes were killed, the second sterile buffer rinse was also plated on PDA. If no microbial growth occurred on the sterile rinse plate, we could assume that the remaining microbes cultured from the homogenized body came from SWD’s digestive tract.

Both the exterior and interior fungal solutions were serially diluted, plated, and incubated at room temperature for 1 – 2 weeks. Plates were assessed for the presence or absence of Botrytis and Cladosporium. We also calculated the resulting colony forming units / mL (CFUs) per fly based on the average number of colonies in the serial dilution containing a countable colony range (2 – 80 spores), which provided a measure of fungal density. CFU data was analyzed separately for the Botrytis and Cladosporium assays using an ANOVA with the nlme package in R, with plate type (interior or exterior fungi), time point, and the plate by time interaction included as fixed effects.

Objective 1B: Evaluate SWD’s vectoring ability and persistence of fungal propagules

Concurrent vectoring assays were conducted to correlate SWD fungal propagules acquisition with the ability to vector pathogens to new substrates. For each replicate, an additional four SWD (two males and two females) were removed from the same fungal cultures used in Objective 1A at the 0-hour time point and placed onto individual PDA dishes. Each fly was then serially transferred onto fresh PDA at the 24, 48, and 72 hours post exposure time points. If flies were capable of vectoring pathogens, then we expected to see fungal colonies grow on the PDA. Plates were incubated for two weeks and assessed for the presence or absence of Botrytis and Cladosporium. Fungal identifications were morphologically confirmed using characteristics such as spore ontogeny and color as described in Barnett and Hunter 1981.

Objective 2: Survey field-collected SWD for associations with plant pathogenic fungi.

To assess adult SWD fungal associations, field surveys were conducted from 2018 – 2020 using mixed raspberry/blackberry fields at two field sites: the Western Maryland Research and Education Center (Keedysville, MD) and the Wye Research and Education Center (Queenstown, MD). In 2018 and 2019, all field sites were unsprayed. In 2020, the Queenstown field site was sprayed with Jet-Ag (26.5% hydrogen peroxide and 4.9% peroxyacetic acid; a crop sanitizer that is marketed for pre- and post-harvest disease control) as part of a separate field efficacy trial. To minimize the potential effects of these sprays, all 2020 fly collections at Queenstown were timed to occur at least one week after Jet-Ag applications. At the 2020 Keedysville field site, the raspberry field in which we collected SWD was also used for a separate insecticide spray trial; plots were sprayed with Delegate WG, a proprietary insecticide at various concentrations, or water. No fungicides were applied at this field site, and flies were only collected from unsprayed rows at the field margin.

At each field site, between 4 – 10 adult SWD were hand-collected on two or three separate dates between August and October annually using an ethanol sterilized aspirator. Flies were immediately drowned in sterile buffer solution, chilled on ice, and transported to the University of Maryland campus for immediate processing in lab. We evaluated each fly for the presence of fungal spores on both the cuticle and within the gut using the methods described in Objective 1a. Serial dilutions of both the exterior and interior fungi were plated on sterile PDA, incubated at room temperature for two weeks, and monitored for fungal growth. Any fungi that grew were isolated using a flame sterilized pick and cultured using hyphal tipping to ensure that fungal strains were pure and uncontaminated.

In 2020, we also surveyed the raspberry/blackberry fruit fungal community present in our field sites at the time of the SWD collection, generating background information about the prevalence of primary fungal pathogens within these fruiting systems and helping us to infer whether SWD acquires fungal propagules from the raspberry/blackberry plants or from other sources (e.g. non-crop plants). At Queenstown, we performed post-harvest fruit rot evaluations on only one sampling date due to limited fruit availability; all fruit at Queenstown were harvested weekly for a separate field study that evaluated different trellising systems. Among the blackberries harvested from Queenstown, 60 marketable fruit were randomly selected and incubated in humid, bleach-sterilized crisper boxes for one week, at which point we assessed each berry for the presence or absence of fungi. Any fungi detected were cultured for subsequent identification. At Keedysville, fruit availability was not a limiting factor, allowing us to conduct visual surveys of pre-harvest fruit rot infections on both sampling dates. In each row that flies were collected (2 rows during the first collection and 3 rows during the second collection), we randomly selected and assessed 40 berries for fruit rot. Berries were selected from both sides of the row as well as from varying canopy heights and locations (inner and outer canopy). Fruit that had a visible fungal infection were harvested and returned to lab, where fungi were isolated for subsequent identification.

Identification of the fungal isolates from both SWD and the caneberry fruit was conducted using molecular methods. All fungal isolates were initially identified to genus through PCR and DNA sequencing of the ITS region of the fungal genome. To help determine if our fungal isolates were specifically pathogenic to raspberries, a subset of these fungal identifications were pushed to the species level through additional PCR reactions and sequencing of different regions of the fungal genome. These species-level identification efforts focused on four target genera: Botrytis, Cladosporium, Colletotrichum, and Fusarium. We selected these genera because they were commonly detected on SWD, contained known raspberry pathogens, and/or were also isolated from the fruit sampled in 2020.

Objective 3: Evaluate how Botrytis and Cladosporium fruit rot impact SWD egg-laying behavior

Preliminary, no-choice egg-laying assays were conducted in 2018 to evaluate how fruit rot pathogens impact SWD egg-laying behavior. Mated SWD were exposed to raspberry agar media (blended raspberry fruit + water + 2% agar)  that had been inoculated with a Botrytis or Cladosporium spore suspension (100 uL at a concentration of 1.5 x 104 spores / mL) that was either 0, 1, 4, or 7 days old. Varying the age of the spore suspensions allows us to quantify how fungal infection severity could impact egg-laying. As an untreated control, flies were also exposed to non-inoculated raspberry agar plates with the same incubation time points.

All experiments were conducted using SWD that were 4-5 days old. 15 female and 5 male flies were held in egg-laying arenas containing the treated raspberry agar. After 24 hours, plates were frozen to halt egg-laying, and we counted the number of eggs deposited on each plate using a Leica M80 stereomicroscope within one week of freezing plates. Egg-laying activity was quantified as the number of eggs laid per female.

Research results and discussion:

Objective 1

Rates of fungal propagule acquisition were high under no-choice laboratory conditions. In the laboratory vectoring assays (Objective 1b), SWD successfully transmitted both Botrytis and Cladosporium to sterile media through the 72-hour time point (Table 1). SWD from the untreated control (sterile PDA) did not acquire or vector Botrytis and Cladosporium propagules at any time point, confirming that our colony was not contaminated with either fungus at the time of these experiments.

Table 1: Percentage and standard error of SWD that successfully transmitted Botrytis or Cladosporium to sterile media 0, 24, 48, and 72 hours post exposure (Obj. 1b). Sterile PDA was used as an untreated control to confirm that contamination did not occur during the trials. 

We observed no differences in fungal acquisition rates between male and female SWD, so all data on fungal incidence and density over time (Objective 1a) were pooled across sex. For Botrytis, 100% of the flies tested carried exterior fungal propagules through 48 hours, and 91.7 ± 8.3% of flies still carried fungi on their exterior at 72 hours (Table 2). Similarly, 100% of the SWD exposed to Cladosporium scored positive for carrying fungi on their cuticle 48 hours after exposure; by the 72-hour time point, that number dropped to 87.5 ± 8.5%. Although persistence of fungi remained high throughout the study period, the density of fungal propagules accumulated externally decreased over time for both Botrytis (F3,15 = 18.145, P < 0.001; Figure 2) and Cladosporium (F3,15 = 9.215, P = 0.001; Figure 2), a pattern that may stem from grooming behavior commonly observed in SWD and other species of Drosophila.

Table 2: Mean percent and standard error of SWD that had viable interior and exterior fungal propagules 0, 24, 48, and 72 hours after exposure to sporulation Botrytis or Cladosporium.

Rates of fungal acquisition and persistence were lower within the interior (ingested fungi) compared to the exterior of the body (Table 2, Figure 3), suggesting that the exterior cuticle may be more important for flies acquiring and/or transmitting fungal propagules. Total ingested Cladosporium incidence decreased from 91.7% ± 5.3% of SWD surveyed 0 hours post exposure to 68.1% ± 17.3% at the 72-hour time point. In contrast, 87.5% ± 5.6% of flies surveyed 0 hours post exposure were found to carry some amount of Botrytis within their alimentary canal. However, that number decreased to 8.3% ± 5.3% of flies by the 24 hour post exposure time point and remained low for the remainder of the study (Table 2). These differences between Botrytis and Cladosporium likely stem from differences in fungal density, as flies appeared to consume greater quantities of Cladosporium relative to Botrytis (Figure 3).

Overall, interior fungal density tended to be lower that the exterior density. For example, we found that flies carried an average of 4,502.1 ± 1,967.1 Cladosporium CFUs/mL on their cuticle immediately after being removed from the sporulating fungal culture (0-hour timepoint; Figure 2), compared with 734.2 ± 192.1 CFUs/mL within their gut at 0 hours (Figure 3). Overall, the density of interior (ingested) Botrytis (F3,15 = 15.779, P < 0.001) and Cladosporium (F3,15 = 16.896, P < 0.001) decreased over time, which likely reflects spores degrading within the midgut and/or passing through the digestive tract.

Figure 2: Mean number of (A) Botrytis and (B) Cladosporium colony forming units (CFUs) + standard error isolated from the cuticle (exterior fungi) of SWD 0, 24, 48, and 72 hours after exposure to sporulating fungal cultures (N= 6 replicates). Bars that do not share a letter are significantly different at α=0.05. 

Figure 3: Mean number of (A) Botrytis and (B) Cladosporium colony forming units (CFUs) + standard error isolated from the midgut (interior fungi) of SWD 0, 24, 48, and 72 hours after exposure to sporulating fungal cultures (N= 6 replicates). Bars that do not share a letter are significantly different at α=0.05. 

Objective 2

Filamentous fungal associations were fairly ubiquitous among field-collected adult SWD. 96% of the adults that we sampled (N=71 SWD pooled across 2018, 2019, and 2020) carried one or more genera of fungi on their body, and many flies (60%, 85%, and 44% in 2018, 2019, and 2020 respectively) had fungi present in/on both their interior and exterior. In total, 39 unique genera of fungi were isolated, although the majority of fungal genera detected occurred relatively infrequently. For example, out of the 30 genera identified in 2020, 18 were isolated from only a single fly.

Many flies carried multiple fungi on their bodies simultaneously; across all sampling years, exterior genus richness ranged from 0 to 6 unique genera isolated per fly and interior richness ranged 0 to 3 genera per fly. In 2018, genus richness increased by 65% between the interior and exterior sampling locations (Figure 4; F1,23 = 25.81, P < 0.001). No significant differences in richness were observed in 2019 (F1,5 = 1.12, P = 0.326), and significant increases in exterior genus richness only occurred at Queenstown in 2020 (F1,37 = 5.31, P = 0.027). The variable patterns in genus richness observed at Queenstown and Keedysville in 2020 may reflect the overall genera richness observed on flies collect at each site; at Queenstown, we isolated 29 unique genera of fungi total across both sampling dates. In contrast, only 10 fungal genera were isolated from Keedysville in 2020; although fewer total genera were collected at this site, the taxa observed occurred more frequently and many were found both internally and externally within flies.

Figure 4: Mean number of fungal genera + standard error (SE) isolated from the interior and exterior surface of field collected SWD in A) 2018, B) 2019, and C) 2020 at Keedysville, MD (KD) and Queenstown, MD (Wye). Data were analyzed separately by year with a mixed model ANOVA that included sampling site, sampling type, and the site by type interaction; significant effects are presented graphically. Within a year, bars that do not share a letter are significantly different by Tukey’s HSD (p<0.05).

Among the fungi identified, Cladosporium was the most frequently isolated genus, occurring on the exterior surface of 33 out the 71 flies that we sampled (Table 3). Other fungi included genera that contain known plant pathogens of caneberries or other crops (e.g. Penicillium, Mucor, Colletotrichum, and Fusarium). Many of the taxa containing known plant pathogens were repeatedly detected on flies that were sampled at both field sites and across multiple sampling dates. Surprisingly, we also isolated fungi that primarily colonize non-crop habitats that include live or decaying wood, soil, indoor structural habitats, or contain known human pathogens.

Table 3: Number of field-collected SWD that were found carrying fungal genera on their cuticle (Exterior) and/or within their digestive tract (Interior). Data were pooled across sampling years and sites, out of 71 flies sampled in total. When possible, fungal genera were categorized by their primary ecological niche. Miscellaneous refers to genera containing species with mixed ecological functions (e.g. contains both plant pathogenic and soil-dwelling species). 19 fungal genera that were only isolated from one fly total were excluded from this table. 

To more fully understand the ecological role of our fungal isolates, we conducted additional PCR and DNA sequencing to achieve species-level identifications in four target genera (Botrytis, Cladosporium, Fusarium, and Colletotrichum). In total, we identified 12 unique species or species complexes, including several known pre-harvest raspberry pathogens as well as fungal species known to cause disease in other fruit and/or non-fruit crops (Table 4). Many of the species identified, including Cladosporium cladosporioides, Cladosporium pseudocladsporoides, and Colletotrichum fioriniae, were detected on multiple flies and across multiple sampling dates and field sites (Table 4). Two of the fly-associated species, Fusarium fujikuroi and Fusarium proliferatum, were also identified from raspberries that were sampled pre-harvest at Keedysville (Table 5).

Table 4: Summary of species-level identifications for fungi isolated from SWD. Each column shows the number of flies with each species of fungi present internally and externally, pooled across field sites and all three sampling years (71 SWD sampled in total). When possible, each species was assigned an ecological role based on previously published studies and pathogenicity testing.

We observed some overlap between the fungi isolated from SWD and fungi isolated from fruit in 2020 (Table 5). For example, five of the fungal genera isolated from raspberry fruit at Keedysville in 2020 (Botrytis, Fusarium, Geotrichum and Mucor) were also found on SWD. Similarly, Gilbertella, the only fungi isolated post-harvest in the Queenstown blackberries, was also predominant in our fly-associated samples.

Table 5: Incidence of fungi isolated pre-harvest from raspberry fruit at Keedysville in 2020 and post-harvest from blackberries at Queenstown in 2020. Columns show the total number of berries sampled at each site (N) and those found to be infected with each fungal taxon.

Objective 3

Preliminary oviposition trials were conducted in 2018. Overall, oviposition rates were highly variably across replicates and may have been influenced by both the fungal treatment and the number of days plates were incubated. In all fungal treatments (Botrytis, Cladosporium, and untreated control), oviposition rates decreased as the incubation time increased, suggesting that incubating the raspberry agar at room temperature reduced its quality as an oviposition substrate for SWD, regardless of whether Botrytis or Cladosporium was present. This, combined with the high variability in oviposition rates, prevented us from determining the extent to which fungal priority impacts host quality for SWD. Media dehydration may have been a confounding factor in these preliminary experiments, as control plates were not inoculated with sterile water prior to incubation; in contrast, additional moisture was introduced into the Botrytis and Cladosporium treatments through the spore suspensions used to inoculate plates. We also ran the oviposition assays in our laboratory instead of using a temperature, light, and humidity-controlled growth chamber; fluctuating environmental conditions may have influenced oviposition rates between replicates and contributed to the high levels of variability observed. Research restrictions associated with the covid-19 pandemic, including limited access to laboratory facilities, limited labor, and supply chain interruptions, prevented us from completing these oviposition assays during the project period.

Research conclusions:

SWD weakly associates with a broad filamentous fungal community. Across three years, we identified 39 unique fungal genera from 71 field collected adult SWD. However, most fungal associates were infrequently detected, with 49% of genera detected only on a single fly. Many of these rarer taxa primarily colonize non-caneberry hosts and were only detected on the exterior region of the fly’s body. Even the more commonly occurring genera, Cladosporium, Fusarium, and Mucor, were found on less than 50% of the flies surveyed. This suggests that SWD’s association with filamentous fungi is largely unintentional and that flies do not seek out particular fungal taxa. Instead, it is likely that flies accidentally acquire fungal propagules on the exterior of their body as they move between crop and non-crop resources. This acquisition may occur when flies land on infected substrates to rest between flights or while assessing fruit quality; female Drosophila spp. often rely on a combination of olfactory, tactile, and gustatory cues to select appropriate egg-laying sites, necessitating direct contact with their host plant.

These unintentional associations still have the potential to affect the raspberry fruit rot community. Species-level identifications of field-isolated fungi indicate that at least some of SWD’s fungal associates can facilitate pre-harvest disease in raspberries. Although they associate with SWD at a low incidence, these pathogenic species were repeatedly detected across multiple field sites and sampling periods, and laboratory vectoring assays indicate that fungal propagules can potentially persist on a fly’s body for a prolonged period of time. Given that flies can transmit fungi to sterile media under no-choice laboratory conditions, it is therefore plausible that SWD increases raspberry fruit rot disease incidence by acting as a vector. Beyond vectoring, SWD oviposition wounds and feeding may also impact disease severity. Indeed, pathogenicity assays with Cladosporium have demonstrated that wounds facilitate disease development, suggesting that SWD may play a role in Cladosporium fruit rot’s epidemiology.

To the best of our knowledge, this work is the first to demonstrate a possible vectoring relationship between SWD and raspberry fruit rot fungi. Additional work will be necessary to fully understand the extent to which these laboratory vectoring relationships persist under field conditions. However, advancing our understanding these relationships will be critical for developing sustainable integrated pest management programs in caneberries. SWD and fungal pathogens are currently treated as separate management issues within caneberry production. However, there is a growing body of work suggesting that these key pests can interact with and influence one another. Accounting for these interactions will help growers to develop management programs that more efficiently target both insects and fungal pathogens.

Participation Summary

Education & Outreach Activities and Participation Summary

12 Webinars / talks / presentations

Participation Summary:

120 Farmers
6 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:

Preliminary results from this study were presented at three national scientific meetings in 2018: the national meeting of the American Chemical Society in Boston (invited talk), the joint annual meeting of the Entomological Society of America and the Entomological Societies of Canada and British Columbia, and the International IPM Symposium in Baltimore (invited talk). Results were also presented through an invited seminar with the Entomological Society of Washington in 2018. One extension presentation related to this work was also given at the Bay Area fruit school meeting in Queenstown, MD (February 2018) and reached approximately 50 stakeholders, including growers, extension agents, and other University of Maryland Researchers.

In 2019, results were also presented at two additional invited talks (the national meeting of the American Chemical Society in San Diego and at a Department of Entomology seminar at Michigan State University), as well as one regional meeting (the annual meeting for the Eastern Branch of the Entomological Society of America). Results from this study were also presented at the 2019 Graduate Student Research Day, a multi-disciplinary event hosted by the University of Maryland Graduate School.

In 2020, we originally planned to present work related to this study at two invited talks (2020 International Congress of Entomology and the 2020 meeting for the Eastern Branch of the Entomological Society of America); however, these events were postponed and cancelled respectively due to the Covid-19 pandemic. Instead, results related to this study were presented to various audiences through virtual meeting formats, including a YouTube video that was produced in lieu of a twilight field tour (70 views as of 6/21/21) and two presentations at the virtual meeting for the Entomological Society of America. In spring 2021, results from this study were also presented virtually at the eastern branch meeting for the Entomological Society of America.

Project Outcomes

3 Grants applied for that built upon this project
1 Grant received that built upon this project
$30,000.00 Dollar amount of grant received that built upon this project
1 New working collaboration
Project outcomes:

The results from this project will guide the development of more integrated insect and pathogen management programs in mid-Atlantic fruit production. In general, management decisions for SWD and fruit rot fungi such as Botrytis are made independently from one another. However, early results from this study indicate that adult SWD have the potential to interact with and influence fungal disease patterns. We found that under no choice conditions, SWD acquire, retain, and vector both Botrytis and Cladosporium at high rates. Our field surveys also indicate that adult flies associate with a diverse fungal fauna that includes Cladosporium and several other potentially pathogenic genera of fungi. Future work will continue to advance our understanding about how these vectoring relationships impact fruit quality under more field-realistic conditions.

An improved understanding of the interactions between SWD, Botrytis, and Cladosporium could guide the development of more targeted pest management tactics, as has been seen in other systems. For example, a recent study indicates that grape sour rot disease is most effectively controlled through spray programs that combine antimicrobial products with insecticides that target Drosophila spp. If a similar relationship exists in raspberries, growers may be able to minimize damage from Botrytis and Cladosporium through more careful control of its insect disease vector. This may include integrating pesticide and fungicide applications. In organic production (where pesticide options are more limited), the integration of cultural control tactics, including exclusion netting, sanitation, and habitat manipulation could also reduce SWD populations, consequentially contributing to pathogen management. In addition to increasing profits (through a reduction of Botrytis or Cladosporium-mediated crop loss), such integrated management programs may also reduce reliance on fungicides for Botrytis and Cladosporium, further enhancing agricultural sustainability.

Knowledge Gained:

The process of designing and planning this project has greatly improved my awareness of the importance of taking an integrated approach to agricultural pest management. When I began my graduate degree, my research interests focused primarily on insect pests in agriculture. However, through my course work, conversations with growers, and conducting literature searches for this project, I gained an appreciation for the broad array of pests that growers must contend with, including weeds, insects and plant diseases. Tailoring management efforts to one “pest” or one category of pest is not a sustainable approach to agriculture, as these organisms frequently interact and influence one another. Taking the time to understand these interactions can guide the development of holistic sustainable approaches.

I plan to pursue a career in applied entomology, with my research focused on improving sustainable pest management options within agriculture. The skills that I acquired through this project have contributed to my professional development and will help me move forward in my career. For example, I supervised six undergraduate students in research tasks related to this project over the course of the past three years. Through this work, I have gained experience in both mentoring undergraduate students and managing a project’s workflow. I also expanded my skills in experimental design and trouble-shooting while designing and implementing behavioral assays for SWD. Results from this study have been presented at several scientific conferences, giving me experience in public speaking and professional science communication. Moving forward, I will also continue to expand these skills through writing an additional extension article as well as a peer reviewed publication (anticipated submission late July 2021). I also plan to present results related to this work at the upcoming national Entomological Society of America Meeting in November 2021. 

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.