Progress report for GNE21-248
Project Information
Sour rot is an economically important disease of vineyards in wet and humid environments. Physical injury to berries is considered an essential factor for disease incidence as it may provide a pathway for microbes known to cause sour rot. Moreover, Drosophila fruit flies and other common insects are suspected to act as vectors of sour rot-related microbes and/or wounding agents near harvest season in vineyards. One of our objectives was to assess whether yellow jacket and grape berry moth damage to berries interact with Drosophila melanogaster to increase sour rot disease. To address this, we conducted a semi-field experiment in the Vignole vineyard in Cornell Agritech and presented research results at the CRAVE (Cornell Recent Advancement in Viticulture and Enology) conference and the Long Island meeting. We found that yellow jackets in the presence of Drosophila melanogaster significantly increase sour rot severity mediated through injuries they cause to berries. This information will be instrumental in developing holistic management practices for sour rot in vineyards. Due to the poor establishment of grape berry moth larvae in selected bagged clusters in vineyards, we could not thoroughly understand the impact of grape berry moth on sour rot.
1. Understand the role of odors in mediating interactions between sour rot and Drosophila
1A. Assess D. melanogaster behavior in response to odors produced from inoculated and non-inoculated berries
We hypothesize that sour rot-associated microbes alter the volatile composition of berry tissue and thus alter D. melanogaster behavior. We will inoculate grapes with a sour rot-associated microbes and conduct choice bioassays to observe the behavior of D. melanogaster over time. We will also evaluate D. melanogaster preference for odors from treated berries with and without larvae.
1B. Assess volatile profiles of inoculated and non-inoculated berries
We hypothesize that disease associated microbes alter berry odors that D. melanogaster uses to distinguish amongst berries. We will collect volatiles from berries inoculated with yeast and bacteria with or without larvae in laboratory assays and conduct volatile analysis using GCMS. These data will provide insights into possible changes in the volatile profile of berries associated with differences in behavior uncovered in objective 1A.
2. Evaluate whether suzukii infestation facilitates subsequent D. melanogaster infestation
2A. Quantify D. melanogaster behavior when provided with berries with larvae or probing marks by D. suzukii
D. suzukii is active in vineyards earlier than D. melanogaster as they prefer to lay eggs in ripening and ripe berries. We hypothesize that this early berry damage caused by D. suzukii activity allows D. melanogaster to lay eggs in fruit more easily, thereby aggravating sour rot risk. We will evaluate whether D. suzukii activity affect the foraging and oviposition of D. melanogaster through video-recordings of fly behaviors.
2B. Evaluate facilitation of D. melanogaster by D. suzukii in berries and its impact on sour rot incidence and severity in the field
We will specifically test whether injuries and cues left by D. suzukii at different time points increase the likelihood that D. melanogaster lays eggs and how that differentially affects sour rot development in field trial.
3. Assess whether damage from yellow jackets and grape berry moth (GBM) interacts with Drosophila and sour rot development
As grapes ripen, yellow jackets forage on berries for sugars and pulp. GBM larvae feed inside berries during late summer. We hypothesize a synergistic interaction occurs amongst yellow jackets and GBM berry damage, Drosophila flies, and microbes, causing increased sour rot disease. We will conduct a field trial with enclosed grape clusters in mesh bags to manipulate the presence of D. melanogaster, GBM, yellow jackets, and causal microbes.
Sour rot is an important disease of wine grapes and poses a significant economic threat to grape production, especially in the eastern US, where summer weather is warm and wet. Sour rot reduces the quality of wine grapes by imparting a pungent vinegar smell to the juice. Sour rot also reduces total yield by either disintegrating or rendering the cluster unusable and necessitates high labor costs at harvest associated with the need to sort out diseased berries.
The disease is caused by a complex multi-trophic interaction of insects like Drosophila melanogaster, microbes like acetic acid bacteria and yeast, and grape berries. Drosophila flies are responsible for vectoring sour rot-associated microbes present on their body surface and gut to healthy berries. They are attracted to, and thus disease is often facilitated by, cracks in berry skin caused by mechanical forces, birds, and very likely, insects. Additionally, Drosophila larvae feeding in the berry can facilitate disintegration and aggravate disease symptoms. Moreover, the invasive vinegar fly D. suzukii becomes abundant in vineyards at about the same time grapes begin to ripen and may initiate damage to healthy berries. D. suzukii was found to emerge from berries at a mild-rot stage, and before D. melanogaster emergence, suggesting that D. suzukii oviposition precedes D. melanogaster egg-laying. D. suzukii oviposits by drilling through the fruit surface with their serrated ovipositor. This, plus tunneling by D. suzukii larvae, may provide greater access to fruit for adult D.melanogaster, which lack a serrated ovipositor, resulting in more sour rot disease. Yellowjackets and grape berry moths may also play a role in sour rot ecology via their ability to cause both berry injury and transfer microbes from diseased to healthy clusters. Overall, insects appear to play an important role in sour rot etiology.
Currently, sour rot management relies on weekly insecticide applications, targeting Drosophila fruit flies, coupled with antimicrobial pesticides starting several weeks before harvest. Such heavy reliance on insecticides is not sustainable and has resulted in documented emergence of insecticide resistance in D. melanogaster to several different classes of insecticides.
Thus, the increased resistance problems make imperative the development of suitable IPM tactics for monitoring and managing Drosophila fruit flies below the economic threshold level in vineyards. Growers need to know the activity of insects, mainly D. melanogaster and its interaction with other insects and their contribution to sour rot in vineyards before making any pest management decisions.
The overall purpose of this study is to better elucidate insects’ role as potential vectors and agents of disease, document the classes of insect interactions that facilitate and exacerbate sour rot, and elucidate insect behavior in response to rot-associated cues to improve integrated management strategies for sour rot.
Research
Fly stocks
Fly stocks will be reared using a standard cornmeal-based Drosophila diet at Cornell AgriTech, Geneva, NY, in a climate‐controlled chamber at 25 ± 1 °C, 16:8 h light: dark photoperiod, and 60 ± 5% humidity. Chambers will also be used for behavioral experiments. Newly enclosed flies will be moved to new media every 48 h and allowed to mature for 5–7 days before testing. Male and female flies will be tested separately.
Grape inoculation
A suspension containing yeast (Metschnikowia pulcherrima) and bacteria (Gluconobacter oxydans), causal microbes of sour rot, will be used to inoculate berries. M. pulcherrima will be maintained on PDA and Gluconobacter oxydans on LPGA media at 4°C. Bacteria and yeast isolate identity will be confirmed with Sanger sequencing using PCR amplicons of 16S ribosomal RNA bacterial gene regions for bacteria and ITS/5.8S rRNA gene regions for yeast (1).
Grapes from a supermarket will be used for all experiments following standard practice (2) with slight modifications. Three berries will be used for each experimental unit. After surface sterilization, berries will be wounded with a sterile toothpick inserted into the berry center. The berries will be inoculated by pipetting 50 µl of a microbial suspension into the wound and incubated at 24°C with 12-h light/dark photocycle for five to eight days.
- Understand the role of odors in mediating interactions between sour rot and Drosophila fruit flies.
Choice bioassay
Two gated traps (allow entry of fly but prevent escape), each fitted with a cut 0.7mL centrifuge tube, will be used. The traps will be wrapped with foil to remove confounding visual cues. Each gated trap will include a deli cup (60ml) with four berries, with one set inoculated with bacteria and yeast suspension and the other sham inoculated as treatments. One female or male D. melanogaster will be released into the arena. Behavioral assays will begin at approximately 10:00 am and run for a 24-hr. period at 22.5 ± 0.5 °C, after which the location of the fly will be recorded. Each cup or beaker will be considered a replicate. We will run 10 replicates per day for three days for a total of 30 replicates.
In a similar experimental setup, the olfactory preference of D. melanogaster between inoculated berries with and without D. melanogaster larvae will also be assessed. After inoculation of all berries, each berry of the treatment with larvae will receive ten D. melanogaster eggs and be incubated for two days to allow eggs to hatch. Choice bioassays as described above will then be performed.
The difference in the proportion of flies that choose either of the treatments in choice bioassay will be analyzed using a Simple Pearson Chi-square test at α = 0.05.
- Evaluate whether infestation by D. suzukii facilitates the subsequent D. melanogaster infestation
Choice bioassay
- Evaluate whether infestation by D. suzukii facilitates the subsequent D. melanogaster infestation
The olfactory preference of female D. melanogaster was compared in response to the berries pre-infested with female D. suzukii and artificially probed berries 24 hours before the actual bioassay. For the pre-infested berry treatment, five lab-reared female D. suzukii were exposed to two sterilized berries with intact pedicel for 24 hours before the actual bioassay (starting 3 pm in the evening) in a 100ml sterilized beaker covered with sterilized mesh. Control treatment consisted of two sterilized berries probed with sterilized needles (on average 5 probings) to make it comparable with the actual treatment that consisted of approximately 5-8 probings from flies. The experimental unit consists of a single female D. melanogaster, and treatments consisted of two berries exposed with five female D. suzukii and two artificially probed (using sterilized needle) berries in a 32-ounce sterilized deli-cup with a transparent lid cover for observing behavior and two adjacent meshed holes (dimension) on a side wall for aeration. The movement of insects was tracked visually and by using Etho Vision where we measured a) the number of times D. melanogaster lands (and b) the time spent walking on pre-infested and artificially probed berries. A similar bioassay was conducted to assess and quantify the behavioral response of normal D. suzukii when provided a choice between berries exposed with five female axenic D. suzukii flies (devoid of gut microbes) and artificially probed berries through visual observation. The visual observation and quantification of choice-making behaviors lasted for one and a half hours after which berries from the choice bioassays were left as it is in the same deli cup. After 24 hours, corresponding berries from treatment and control were kept in a separate 32-ounce deli cup to rear out flies, especially D. melanogaster to evaluate whether there is a difference in the number of flies in each treatment.
The differences in duration of each behavior between each treatment will be analyzed using a linear mixed model. The differences in the number of times fly select each treatment will be analyzed using a generalized linear mixed model with the Poisson distribution in R software version 3.6.3 (4).
Field experiment
At around 15ο Brix, intact, undamaged clusters of the sour rot-susceptible cultivar Vignoles in a research vineyard at Cornell AgriTech will be inoculated with a spray solution containing acetic acid bacteria and yeast. A single cluster of grapes will be exposed to one of four different treatment conditions enclosed in a fine mesh bag: D. suzukii alone, D. melanogaster alone, with both species together, and with no flies. For treatments with both, we will introduce D. melanogaster into the enclosure at different times after the introduction of D. suzukii: 1) D. suzukii and D. melanogaster together 2) D. suzukii infestation followed two days later by D. melanogaster 3) D. suzukii infestation followed three days later by D. melanogaster 4) D. suzukii infestation followed 4 days later by D. melanogaster. We will use six females from each species and ten replicates per treatment. Ten days after flies are introduced, sour rot severity will be measured, and flies will be reared from berries for assessing fly emergence.
- Investigate the interactions among insect damage to berries (yellow jackets, grape berry moth), Drosophila fruit flies, and sour rot
At around 15 Brix, intact, undamaged clusters of Vignoles will be inoculated with a spray solution containing acetic acid bacteria and yeast. Fine mesh bags will be used to assess the impact of damage by insects (grape berry moth, yellowjacket wasps) and mechanical damage by birds, with and without fruit flies in vineyards. Treatments include: 1) undamaged control, 2) mechanical damage to 20% of berries to simulate bird injury, 3) five grape berry moth larvae from our lab colony, 4) two field-collected Eastern Yellow Jacket adults, 5) undamaged clusters followed 7 days later by ten mated female D. melanogaster flies, 6) mechanically damaged berries followed 7days later by D. melanogaster, 7) grape berry moth followed 7days later by D. melanogaster, and 8) yellow jacket adults followed 7days later by D. melanogaster were used for the experiment. Ten days after flies are introduced, clusters will be harvested and evaluated for sour rot incidence and severity.
For objectives 2B) and 3), we will use a linear mixed model to analyze the severity of sour rot among treatments using ‘nlme’ package. Additionally, we will use a generalized linear model with random effect and Poisson distribution using ‘lme4’ package in R version 3.6.3 (4) to analyze the number of flies reared from the treatments.
- Understand the role of odors in mediating interactions between sour rot and Drosophila fruit flies.
Choice bioassay
Two gated traps (allow entry of fly but prevent escape), each fitted with a cut 0.7mL centrifuge tube, will be used. The traps will be wrapped with foil to remove confounding visual cues. Each gated trap will include a deli cup (60ml) with four berries, with one set inoculated with bacteria and yeast suspension and the other sham inoculated as treatments. One female or male D. melanogaster will be released into the arena. Behavioral assays will begin at approximately 10:00 am and run for a 24-hr. period at 22.5 ± 0.5 °C, after which the location of the fly will be recorded. Each cup or beaker will be considered a replicate. We will run 10 replicates per day for three days for a total of 30 replicates.
In a similar experimental setup, the olfactory preference of D. melanogaster between inoculated berries with and without D. melanogaster larvae will also be assessed. After inoculation of all berries, each berry of the treatment with larvae will receive ten D. melanogaster eggs and be incubated for two days to allow eggs to hatch. Choice bioassays as described above will then be performed.
The difference in the proportion of flies that choose either of the treatments in the choice bioassay will be analyzed using a Simple Pearson Chi-square test at α = 0.05.
Results:
At a significance level of 0.05, we can conclude that the preference of female D. melanogaster in response to microbes-inoculated berries is significantly different from the preference in response to non-inoculated injured (sham) berries. (Pearson chi-square = 10, df= 1, p-value = 0.002). However, no difference in female D. melanogaster preference was observed when subjected to treatments for inoculated berries with and without D. melanogaster larvae (Pearson chi-square = 1.07, df= 1, p-value = 0.30).
- Evaluate whether infestation by D. suzukii facilitates the subsequent D. melanogaster infestation
The olfactory preference of female D. melanogaster was compared in response to the berries pre-infested with female D. suzukii and artificially probed berries 24 hours before the actual bioassay. For the pre-infested berry treatment, five lab-reared female D. suzukii were exposed to two sterilized berries with intact pedicel for 24 hours before the actual bioassay (starting 3 pm in the evening) in a 100ml sterilized beaker covered with sterilized mesh. Control treatment consisted of two sterilized berries probed with sterilized needles (on average 5 probings) to make it comparable with the actual treatment that consisted of approximately 5-8 probings from flies. The experimental unit consists of a single female D. melanogaster, and treatments consisted of two berries exposed with five female D. suzukii and two artificially probed (using sterilized needle) berries in a 32-ounce sterilized deli-cup with a transparent lid cover for observing behavior and two adjacent meshed holes (dimension) on a side wall for aeration. The movement of insects was tracked visually and by using Etho Vision where we measured a) the number of times D. melanogaster lands (and b) the time spent walking on pre-infested and artificially probed berries. A similar bioassay was conducted to assess and quantify the behavioral response of normal D. suzukii when provided a choice between berries exposed with five female axenic D. suzukii flies (devoid of gut microbes) and artificially probed berries through visual observation. The visual observation and quantification of choice-making behaviors lasted for one and a half hours after which berries from the choice bioassays were left as it is in the same deli cup. After 24 hours, corresponding berries from treatment and control were kept in a separate 32-ounce deli cup to rear out flies, especially D. melanogaster to evaluate whether there is a difference in the number of flies in each treatment.
The differences in duration of each behavior between each treatment will be analyzed using a linear mixed model. The differences in the number of times fly select each treatment will be analyzed using a generalized linear mixed model with the Poisson distribution in R software version 3.6.3 (4).
General result:
Twelve replicated bioassays were conducted to test whether the normal D. melanogaster prefers to land onto and spend more time on the berries pre-infested by five female lab-reared D. suzukii. Looking at the heatmap from Etho Vision, we can vaguely state that lab-reared D. melanogaster prefers to spend more often in berries that are pre-infested or probed by normal D. suzukii (Objective 2 figures: Figure 1).
Twenty replicated bioassays were conducted to quantify the landing behaviors of D. melanogaster in response to the normal fly-probed and mechanically probed berries, D. melanogaster spent more time and frequently landed on normal fly-probed berries (36.79 ± 7.86 seconds, number of times landed = 33) compared to mechanically probed berries (3.85 ± 1.14 seconds, number of times landed = 13). D. melanogaster female was allowed to lay eggs in a choice bioassay trial set up for quantification study after one and half hours of visual observation. Then, flies were reared out in a separate deli cups. We did not observe a difference in the number of reared D. melanogaster adults between axenic fly-probed (0.25 ± 0.12) and mechanically probed (0.13 ±0.76) berries (Objective 2 figures: Figure 2). A similar result was found between normal fly-probed (0.25 ± 0.12) and mechanically probed berries (0.13 ± 0.06) (Objective 2 figures: Figure 3).
Normal D. melanogaster females spent almost equal time in axenic fly-probed berries (24.82 ± 4.2 seconds) and mechanically probed berries (23.47 ± 3.3 seconds) (Objective 2 figures: Figure 4).
Field experiment
A semi-field experiment was conducted to test whether pre-infestation of grape berries by D. suzukii at a different time increases sour rot severity by facilitating D. melanogaster to lay a high number of eggs compared to negative control and positive control.
At around 15ο Brix, intact clusters of “Vignoles”, a sour rot susceptible cultivar, were inoculated with a spray solution containing acetic acid bacteria and yeast in a vineyard at Cornell Agritech Research North (GPS). A single cluster of grapes was randomly assigned to one of the following treatments. The treatments included: 1) D. suzukii and D. melanogaster together 2) D. suzukii infestation followed two days later by D. melanogaster 3) D. suzukii infestation followed three days later by D. melanogaster 4) Berries with microbes only (positive control) 5) Berries without any treatment (negative control) 6) Berries with D. melanogaster only 7) Berries with D. suzukii only. We introduced D. melanogaster into the bag enclosure at different times after the introduction of D. suzukii. We used ten (four male and six female) Drosophila species in respective treatments, and we had ten replicated treatments. Ten days after flies were introduced, sour rot severity was measured, and flies were reared from berries to assess fly emergence.
Results:
A significant difference in sour rot percentage was observed between the negative control treatment and the D. suzukii-only treatment (Z= -3.14, P = 0.03). Although statistically insignificant, sour rot percentage in the negative control treatment and the treatment exposed with D. melanogaster two days after exposure to D. suzukii was nearly statistically significant (Z= -2.94, P = 0.05) (Objective 2 figures: Figure 5).
Objective 3. Investigate the interactions among insect damage to berries (yellow jackets, grape berry moth), Drosophila fruit flies, and sour rot: For the experiment, we selected healthy, undamaged grape clusters in vineyards in Cornell Agritech, Research North at Vignole vineyards. Each cluster was bagged after spraying sour rot microbes suspension. Four different levels of injury treatment were (1) Control, (2) Yellow Jackets, (3) Grape Berry moth (4) Mechanical damage, where each level was allowed to interact in the presence or absence of Drosophila melanogaster flies. Eight different enclosed clusters were exposed to different treatments in 10 replicates.
Results: As a result, we did not observe much impact of grape berry moth on sour rot severity. However, when berries were subjected to treatments such as yellow jackets and mechanical damage, disease severity increased, mainly in the presence of Drosophila melanogaster compared to undamaged control and treatments without flies from both years (2021 and 2022) studies (Objective3 figures: Figure. 1). Similarly, when we reared out flies from the bagged clusters subjected to injury and Drosophila fruit fly treatments in a deli-cup in the laboratory, more adult flies emerged from the berries (Objective3 figures: Figure. 2 ) that were subjected to yellow jackets and mechanical damage in the presence of Drosophila fruitflies. The result from the rearing study suggests that mechanical injury and injury by yellow jackets facilitates Drosophila fruit fly oviposition, resulting in more number flies, leading to a higher sour rot percentage compared to the control. We could not understand the role of the grape berry moth in sour rot due to the poor establishment of grape berry moth larvae in clusters in vineyards.
The first-year study somewhat elucidated the role of multiple insects in sour rot etiology, providing new insights into sustainable disease management strategies. The second-year study showed a similar trend as the first-year study. Although GBM larvae were established in the second-year study, we did not find a significant difference in sour rot severity and fly number in the presence and in absence of Drosophila fruit flies. The graphs showing the final result of this trial are attached in the add media folder that states Objective3 figures.
Education & Outreach Activities and Participation Summary
Participation Summary:
We have communicated results to growers, extension educators, and other relevant stakeholders through newsletter articles that can be open-accessed in Appellation Cornell. Some of the results have been presented among growers-oriented and stakeholders-oriented seminars such as the Long Island Fruit and vegetable updates and national conferences such as the Entomological Society of America. Similarly, we have extended the information to growers and stakeholders through platforms like Twitter sharing the newsletter that includes some of the results related to the work from the SARE Grant. Videos relating to the background and a few results associated with the objectives outlined in the proposal have been submitted to the American Journal of Enology and Viticulture and the storyboard is currently uder review.
Project Outcomes
From our first and second-year trials to investigate the third project objective, we learned that injuries are a very important component of sour rot disease, and yellow jackets- apart from creating annoyance among growers are responsible for creating injuries in berries that facilitate sour rot. From the result, so far, we can confirm that growers need to be concerned about potential berry injuries and adopt yellow jacket management in vineyards for holistic management of the disease.
While conducting an experiment to investigate our third objective, we empirically knew that injuries play a major role in sour rot and the agents of injuries could be as large as mechanical damage (that mimic bird damage or hail damage) or yellow jackets that were captured from vineyards. Despite having sour rot microbes in all the tested clusters, the presence of Drosophila melanogaster only did not cause sour rot at visual and olfactory detection levels at least from our one-year study. This elucidates that there is a need to manage birds, hail, or yellow jacket injuries in vineyards to manage the disease in a sustainable way. Although in our first-year study, we did not get a good establishment of grape berry moth larvae into the berries to be able to understand the role of injury by grape berry moth (GBM) in sour rot in the presence and absence of Drosophila fruit flies. We changed the protocol for deploying GBM larvae where we deployed second instar larvae which caused damage in berries and established well. However, based on the result, we understood that GBM in the presence of Drosophila melanogaster did not produce a similar level of sour rot as we observed in other treatments.
I am interested in solving growers' problems by understanding the pest behavior that would facilitate the development of sustainable agricultural pest management.
While understanding the role of insects in sour rot disease etiology, there was a lack of establishment of grape berry moth larvae despite our efforts to keep them active on the bagged grape clusters. We have not applied for any research grants yet to build upon this project, but we look forward to understanding the role of grape berry moth in sour rot disease in controlled conditions. We developed a better idea of capturing yellow jackets in vineyards after several hit-and-trial processes that we don't have to face in follow-up experiments. In addition, we learned to key them out to make sure we know which species we are using in the bagged trial. During the course of the bagged experiment, we observed different types of yellow jackets belonging to different genera and species. Therefore, we think it is important to address injuries specific to specific yellow jacket species that result in sour rot. Similarly, as a future direction, we can assess the microbes community in yellow jackets that further help to understand the yellow jacket's role as a vector in sour rot disease etiology in vineyards.
Similarly, we changed the protocol for grape berry moth deployment in the bagged experiment. In the first year of the study, we deployed grape berry moth larvae in a slice of commercial berry which did not establish well, however, in the second year, we deployed larvae directly on the experimental grape cluster and got good establishment in berries to be able to pursue the study.
Information Products
- ASSESSING ROLE OF INSECTS IN SOUR ROT DISEASE ETIOLOGY IN VINEYARDS (Conference/Presentation Material)