In this study, I will use a combination of field and laboratory-based approaches to evaluate how interactions between Botrytis and Cladosporium fruit rots and spotted-wing drosophila (SWD; Drosophila suzukii) impact pest and disease dynamics in primocane red raspberries. Specific objectives are to:
- Evaluate the ability of SWD to acquire and transmit Botrytis or Cladosporium propagules under laboratory conditions. These experiments will provide a proof-of-concept for SWD’s vectoring ability and will justify future, field-based experiments to further study SWD’s impact on disease epidemiology.
- Evaluate how Botrytis and Cladosporium fruit rot impact SWD feeding and oviposition decisions. Understanding these behavioral impacts will help us understand how frequently adult flies encounter and potentially acquire Botrytis or Cladosporium propagules under field conditions.
- Survey field-collected SWD for associations with plant pathogenic fungi. This will allow us to determine the frequency and mechanism by which adult SWD acquire fungal propagules under field conditions
The purpose of this project is to evaluate how spotted-wing drosophila (SWD) impacts the incidence and severity 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. Fall-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 and fungal pathogens.
Currently, pest management in raspberries primarily focuses on two organisms: SWD, an invasive fruit fly, and Botrytis cinerea, the causal agent of grey mold. Cladosporium fruit rot, caused by the Cladosporium cladosporioides species complex may also drive mid-Atlantic raspberry crop loss. Cladosporium is generally considered a minor post-harvest pathogen of ripe or overripe fruit. However, recent surveys in Maryland raspberries reported pre-harvest infection rates as high as 30%, suggesting that Cladosporium may have a more significant economic impact than previously believed.
Previous studies have 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 disease incidence and severity. An epidemiological link between SWD and Botrytis or Cladosporium could increase pre-harvest fruit rot incidence and severity, consequentially reducing marketable yield, as is seen in other systems. In raspberries, interactions with SWD could partially account for the recently observed increase in pre-harvest Cladosporium 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
Objective 1: Evaluate the ability of SWD to acquire and transmit Botrytis or Cladosporium propagules under laboratory conditions.
Using field-isolated fungi, we quantified spotted-wing drosophila’s (SWD) 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 PDA plates that were either (1) inoculated with 200 uL of a Botrytis cinerea spore suspension, (2) inoculated with 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 directly 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 Objective 1A and 1B. We are still in the process of completing these no choice assays, but intend to ultimately complete six replicates for each treatment.
Objective 1A: Quantification of gut and cuticle propagule accumulation
We quantified the density of Botrytis and Cladosporium propagules accumulated by SWD on two regions of their body: externally and within the alimentary canal (ingested fungi). Propagule quantification was performed four time points 0, 24, 48, and 72 hours after exposure to fungal cultures. Therefore, we could determine the mechanism by which SWD acquire fungal propagules and quantify how long propagules persist. We examined a total of four flies (2 male and 2 female SWD) for the presence and density of fungal propagules at each time point. Individual flies within a time point were treated as subsamples and pooled for the purposes of data analysis.
For the “0-hour post exposure” time point, flies were directly removed from the fungal culture plate and transferred into 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. At that point, we removed an additional 4 SWD for the “24-hour post exposure” time point and again transferred the remaining flies to a new sterile PDA plate. This process was repeated twice more to generate the 48 and 72 hour post exposure time points.
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 body. Each fly was then transferred to new microcentrifuge tube and surface-sterilized in 95% ethanol for five minutes, followed by two rinses in a sterile buffer solution. The flies were then transferred into a fresh tube containing buffer solution and ground using a sterile pestle. To confirm that all surface-dwelling microbes were killed, the second 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 alimentary canal.
Both the “spore wash” (external fungi) and “whole body homogenate” (ingested fungi) solutions were serially diluted, plated, and incubated at room temperature for 1 – 2 weeks, when the presence or absence of Botrytis and Cladosporium was assessed. We also calculated the 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). CFU data from the Cladosporium assays were analyzed using an ANOVA with the lme4 package in R, with plate type (internal or external fungi), time point, and the plate by time interaction included as fixed effects.
Objective 1B: Evaluate SWD’s vectoring ability and persistence to artificial media
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 (2 males and 2 females) were removed from the same fungal cultures used for 1A 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, we expected fungal colony growth 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: Evaluate how Botrytis and Cladosporium fruit rot impact SWD feeding and oviposition decisions.
We conducted preliminary, no-choice oviposition assays to evaluate how fruit rot pathogens impact SWD oviposition behavior. Mated SWD were exposed to raspberry agar media 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. By varying the age of the spore suspensions, we quantified how fungal infection severity could impact oviposition behavior. As an untreated control, flies were also exposed to non-inoculated raspberry agar plates with the same fungal inoculation time points.
All experiments were conducted using SWD that were 4-5 days old. 15 female and 5 male flies were held in oviposition arenas containing the treated raspberry agar for 24 hours, at which point plates were frozen to halt oviposition activity. We counted the number of eggs deposited on each plate using a Leica M80 stereomicroscope within one week of freezing plates and quantified oviposition activity as the number of eggs laid per female.
Objective 3: Survey field-collected SWD for associations with plant pathogenic fungi.
To assess adult SWD fungal associations, field surveys were conducted from August – October 2018 at two field sites: an unsprayed primocane blackberry patch at Keedysville, MD and an unsprayed primocane red-raspberry patch at Queenstown, MD. At each site, 4 – 10 adult SWD were hand-collected using an ethanol sterilized aspirator. Flies were chilled on ice and transported to the lab in College Park, MD for immediate processing.
We evaluated each fly for the presence of fungal spores on both the cuticle and within the gut using methods similar to those described in Objective 1a. Serial dilutions of both the “spore wash” and “whole body homogenate” were plated on sterile PDA, incubated at room temperature for two weeks, and monitored for fungal growth. Any fungi that emerged were isolated using a flame sterilized pick and hyphal tipped to ensure fungal strains were pure and uncontaminated. We are currently purifying and morphologically identifying each fungal strain to the genus level, and plan to use PCR analysis to obtain species level identifications this upcoming spring.
Overall rates of fungal propagules acquisition and vectoring persistence were high under no-choice laboratory conditions. When exposed to sporulating Botrytis or Cladosporium cultures, adult flies acquired fungal propagules on their cuticle as well as within their alimentary canal (Objective 1a; Figures 1-2). In concurrent vectoring assays (Objective 1b), SWD also demonstrated an ability to vector 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 fungi at the time of these experiments.
We observed no significant differences in vectoring ability between male and female SWD, so all data on fungal propagules incidence and density were pooled between sexes. 100% of SWD exposed to Cladosporium scored positive for carrying propagules on their cuticle through the 48 hours post exposure time point; by the 72 hour time point, that number dropped to 87.5 ± 8.5% (Figure 1). Similarly, the percentage of SWD carrying Cladosporium within their alimentary canal ranged from 91.7 ± 5.2% at 0 hours post exposure to 70.8 ± 16.4% at 72 hours post exposure (Figure 1). Cladosporium density and persistence was highest on the cuticle of SWD (Figure 1), suggesting that this may be the primary mechanism by which flies transmit fungal propagules. For example, 0 hours after exposure, SWD carried an average of 4,502.1 ± 1,967.1 Cladosporium CFUs/mL on their cuticle, compared with 734.2 ± 192.1 CFUs/mL within their gut (Figure 1). We observed significant differences in the density of external and internal Cladosporium propagules acquired over time, with the highest fungal loads for both locations seen 0-hours post exposure (Figure 1). The decrease in external CFUs over time likely stems from grooming behavior commonly observed in SWD and other Drosophila. Similarly, the decrease in internal fungi may reflect either spore degradation and/or passage through the digestive tract.
Botrytis vectoring assays are currently underway. However, preliminary data summaries suggest that flies primarily acquire and retain fungal propagules externally (Figure 2). 100% of the flies surveyed were found to carry Botrytis outside their bodies across all time points (N=3 replicates). In contrast, rates of acquisition within the alimentary canal were low. 83.3% of SWD surveyed scored positive for internal Botrytis at the 0 hour time point. Beyond that initial time point, microbes were only found in one fly at the 72 hour post exposure time point (Figure 2). These trends suggest that SWD feeding on Botrytis could be more limited than what we observed with Cladosporium. Additionally, fungal propagules that were consumed appear to have limited persistence within the alimentary canal.
Oviposition rates were highly variably across replicates and significantly influenced by both the fungal treatment and the number of days plates were incubated. When data were pooled across all time points, we observed a significant decrease in the number of eggs laid per female in plates inoculated with Botrytis. Differences between the control treatment and Cladosporium were not significant but also trended towards reduced oviposition when Cladosporium was present.
As fungal priority increased, oviposition rates decreased across all treatments, including the control raspberry agar (Figure 3). This suggests that incubating raspberry agar at room temperature reduces its host quality for SWD, regardless of whether Botrytis or Cladosporium is 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. We are currently in the process of refining behavioral assays that better address these questions.
In sum, we isolated 117 strains of fungi from SWD. Efforts to purify and identify fungal cultures are on-going. However, preliminary morphological identifications suggest that flies do associate with a diverse fungal community that includes Cladosporium sp.
Education & Outreach Activities and Participation Summary
Preliminary results from this study were presented at two national scientific meetings in 2018: an invited talk at the national meeting of the American Chemical Society in Boston and a submitted talk in the student competition at the joint annual meeting of the Entomological Society of America and the Entomological Societies of Canada and British Columbia.
An extension presentation related to this work will be presented at the upcoming Bay Area fruit school meeting in Queenstown, MD (February 2018). Based on previous years attendance, we expect this information to reach 50 growers, extension agents, and other stakeholders.
The results from this project will guide the development of more integrated insect and pathogen management programs in mid-Atlantic fruit production. Currently, management decisions for SWD and fruit rot fungi such as Botrytis are frequently 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. Preliminary field surveys also indicate that adult flies associate with a diverse fungal fauna that includes Cladosporium and several other potentially pathogenic genera of fungi. As this work continues, we will gain a better sense of 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, recent studies indicate 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 for more careful control of SWD. 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.
The process of designing and planning this project has greatly improved my awareness about 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, including weeds, insects, and plant diseases, that growers must contend with. 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 more sustainable approaches.
Through this project, I also gained a number of skills in project design and management that I will continue to use in my career. I gained experience in designing and troubleshooting behavioral assays. I also acquired a number of new skills in plant pathology, including isolating and culturing fungal pathogens, preparing fungal spore suspensions, and single hyphal tipping. As I move forward in this project, I also will gain skills in fungal identification using both morphological and molecular techniques. Early results from this study have been presented at several scientific conferences, giving me experience in public speaking and professional science communication. I will continue to hone these skills through future extension presentations as well as by writing extension publications and newsletters.