Progress report for GNE18-178
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 (Drosophila suzukii, SWD) 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.
- Assess 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.
- 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 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. 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. In raspberries, Cladosporium is currently considered a minor post-harvest pathogen of ripe or overripe fruit. However, 2016 surveys of 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 fungal disease incidence and severity. An epidemiological link between SWD and Botrytis, Cladosporium, or other raspberry pathogens could increase pre-harvest fruit rot incidence and/or severity, consequentially reducing marketable yield. 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 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 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 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. From 2018 – 2019, 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 fungal cultures. Including these time points allowed us to quantify both the rate at which adult flies accumulate fungal propagules as well as the length of time propagules persisted after the initial exposure. At each time point, four flies (two male and two female SWD) were individually analyzed for the presence and density of fungal propagules on two regions of their body: externally and within the alimentary canal (ingested fungi). 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). At that point, we removed an additional four SWD for the “24-hour” time point and again transferred the remaining flies to a new sterile PDA plate (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.’
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 external fungi did not contaminate isolations from the interior of the fly. 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. 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, which provided a measure of fungal density. CFU data was analyzed separately for the Botrytis and Cladosporium assays 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 (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: Evaluate how Botrytis and Cladosporium fruit rot impact SWD oviposition behavior
Preliminary, no-choice oviposition assays were conducted in 2018 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. Varying the age of the spore suspensions allows us to quantify 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 incubation 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. After 24 hours, plates were frozen to halt oviposition activity, and we counted the number of eggs deposited on each plate using a Leica M80 stereomicroscope within one week of freezing plates. Oviposition activity was quantified 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 in 2018, 2019, and 2020 at two field sites: a raspberry/blackberry patch at Keedysville, MD and a raspberry/blackberry patch at 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 sterilant 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 unsprayed. 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 annually using an ethanol sterilized aspirator. Flies were chilled on ice and transported to College Park for immediate processing. 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 “external fungi” and the “ingested fungi” 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 that fungal strains were pure and uncontaminated.
In 2020, we also surveyed the raspberry/blackberry fruit community present in our field sites at the time of the SWD collection. This data will provide important background information about the prevalence of primary fungal pathogens and will help us to infer whether SWD acquires fungal propagules from the caneberry 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 (interior and exterior canopy). Fruit that had a visible fungal infection were harvested and returned to lab, where fungi were isolated for subsequent identification.
Identification of 2020 fungal isolates from both SWD and caneberry fruit is ongoing and will be completed with molecular methods, including polymerase chain reactions (PCR) and DNA sequencing. DNA was extracted from our 2018 fungal samples using a DNA extraction buffer recipe described in Chi et al. 2009. Due to issues consistently obtaining quality DNA through the Chi et al. protocol, DNA extractions for both the 2019 and 2020 samples were instead performed using the Zymo Bacterial-Fungal Miniprep kit and following the manufacturer’s instructions. Fungal isolates were molecularly identified to genus using the ITS region of the fungal genome. 25 uL PCR reactions were carried out using Promega GoTaq (2018 samples) or Apex Red (2019 and 2020 samples) Master Mix and 0.1 uM ITS1/ITS4 primer; samples were then sequenced using the Sanger sequencing method, and genus level identifications were performed using various online databases (e.g. NCBI Blast, UNITE, and Mycobank).
To determine if our fungal isolates are specifically pathogenic to raspberries, identification to the species level will be necessary. Species-level identifications will require additional PCR reactions and sequencing of different regions of the fungal genome, with specific regions varying between fungal genera. Fungal isolates assigned to a genus that contains known plant pathogens will be identified to species by sequencing additional gene regions. Based on the results of the species level identifications, pathogenicity testing in raspberry fruit will also be conducted for select fungal isolates. Appropriate primers for key fungal taxa (Cladosporium, Botrytis, Mucor, Penicillium, Aspergillus, Fusarium, and Colletotrichum) have been identified and purchased, and we anticipate completing species-level identifications within the next month.
Overall rates of fungal propagule acquisition and 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 differences in vectoring ability between male and female SWD, so all data on fungal propagules incidence and density were pooled across sex. 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 for Botrytis, 100% of all flies tested scored positive for carrying fungal propagules on their cuticle through the 72 hour time point (Figure 1). We observed significant reductions in the density of externally accumulated Botrytis (F3,15 = 18.145, P < 0.001) and Cladosporium (F3,15 = 9.2145, P = 0.001063) over time, a pattern that may stem from grooming behavior commonly observed in SWD and other species of Drosophila.
Rates of fungal acquisition and persistence were lower within the alimentary canal compared to the external body, suggesting that the cuticle may be of more importance for flies acquiring and/or transmitting fungal propagules. For example, 0 hours after exposure to Cladosporium, we found that flies carried an average of 4,502.1 ± 1,967.1 Cladosporium CFUs/mL on their cuticle (Figure 2), compared with 734.2 ± 192.1 CFUs/mL within their gut (Figure 2). We also observed significant decreases in density of both Botrytis (F3,15 = 15.779, P < 0.001) and Cladosporium (F3,15 = 16.896, P < 0.001) over time, which likely reflects spores degrading within the midgut and/or passing through the digestive tract.
Total ingested Cladosporium incidence decreased from 91.6% ± 5.3% of D. suzukii surveyed 0 hours post exposure to 70.8% ± 16.4% 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 remained of the study (Figure 2). This difference most likely stems from differences in fungal density, as flies appeared to consume greater quantities of Cladosporium relative to Botrytis.
Figure 1: Mean number of Cladosporium colony forming units (CFUs) + standard error isolated from the cuticle (external fungi) and alimentary canal (internal fungi) of D. suzukii 0, 24, 48, and 72 hours after exposure to a sporulating C. cladosporioides cultures (N= 6 Replicates). Bars that do not share a letter are significantly different at α=0.05. The numbers above each bar are the percentage of D. suzukii carrying Cladosporium at each time point.)
Figure 2: Mean number of Botrytis colony forming units (CFUs) + standard error isolated from the cuticle (external fungi) and alimentary canal (internal fungi) of D. suzukii 0, 24, 48, and 72 hours after exposure to a sporulating B. cinerea cultures (N= 6 Replicates). Bars that do not share a letter are significantly different at α=0.05. The numbers above each bar are the percentage of D. suzukii carrying Botrytis at each time point.
Table 1: Percentage and standard error of SWD that scored positive for vectoring Botrytis or Cladosporium to sterile media 0, 24, 48, and 72 hours post exposure.
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 after inoculation with fungi (fungal priority). 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.
As we complete these experiments in early 2021, we plan to modify the experimental protocol to account for these and other issues. Proposed experimental modifications will include standardizing the amount of raspberry agar within each petri dish using a serological pipette and treating control raspberry agar plates with water to ensure that standard quantities of moisture are introduced into all treatments. Assays will also only use one female fly to remove potential confounding effects due to aggregation pheromones or aggregation behavior seen in other Drosophila. Oviposition assays will also be repeated using both no-choice and two-choice conditions to determine if egg laying behavior changes when alternative hosts are present.
Pooling data from 2018 and 2019, we identified 23 unique genera of fungi from 31 SWD adults. Identification of our 2020 fungal isolates is ongoing, with genus level identifications expected to be completed by the end of January 2020. In general, higher numbers of fungal isolates were collected from the external body of flies relative to the gut (Table 2), indicating that SWD adults acquire external fungal propagules at relatively high frequencies but do not necessarily consume fungal tissue. It is possible that the reduced fungal density within adult digestive tracts is also due to fungal spores passing through the digestive tract or breaking down within the midgut. Several of the genera that we isolated contain known pre- and post-harvest pathogens, including Cladosporium, Botrytis, Alternaria, Fusarium, Penicillium, and Mucor; many of these potential plant pathogens have also been detected in our preliminary 2020 survey results. However, further identification to the species level will be necessary to determine if our fungal isolates are pathogenic; we expect to complete species-level identifications by the end of February 2020. Surprisingly, we also isolated several genera of wood-associated fungi in 2018, including Schizopyllum, Bjerkandera, and Trametes. Both of our field sites are located near forested areas, so it is possible that this data reflects adult movement between crop and non-crop hosts. However, these wood-associated fungi were not detected in 2019 and have not been detected so far in 2020.
Most fungal genera occurred relatively infrequently. For example, 12 of the 23 genera that we identified were only isolated from one SWD, and 5 genera that we identified were only isolated from two SWD (out of the 31 flies sampled between 2018 – 2019). The most commonly isolated genera (Aspergillus and Cladosporium) were found on less than 50% of the flies surveyed (14 and 13 SWD respectively). These results indicate that SWD has not formed a tight association with any particular fungal taxa. Instead of seeking a particular fungal resource, it is possible that flies unintentionally picked up spores while landing and/or feeding on infected fruit. Collecting background information about the raspberry and blackberry fungal community will help us to better understand these relationships.
Table 2: Number of field-collected SWD that were found carrying fungal genera on their cuticle (External) and/or within their digestive tract (Ingested) in 2018 and 2019. Data were pooled across collection dates and sites, out of 24 flies collected and tested in 2018 and 7 flies collected and tested in 2019. 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)
Education & Outreach Activities and Participation Summary
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; this extension talk was recorded by a local television station and has been made publicly available on YouTube.
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 (40 views as of 1/12/20) and two presentations at the virtual meeting for the Entomological Society of America. This spring, we also plan to publish an extension article that summarizes the key results from this study in University of Maryland Fruit and Vegetable Headline News (estimated readership ~2,500 stakeholders).
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 to sterile media 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. As this work continues, we hope to 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, 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.
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 more 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. Additionally, the results from this study have been used as preliminary data to apply for additional research funding. Working with Dr. Mengjun Hu (Department of Plant Science and Landscape Architecture, University of Maryland) and my advisor, Dr. Kelly. Hamby, I successfully led the writing for a research grant from the Maryland Agricultural Experimental Station (MAES) at UMD. Additionally, I contributed to the writing for a USDA AFRI grant that built off of this project; these experiences have strengthened my grant writing skills and also given me the opportunity to practice initiating and participating in interdisciplinary collaborations. Results from this study have also 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.