Progress report for GNE21-248
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.
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.
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. Following incubation, the presence of sour rot will be assessed based on both (i) a qualitative rating of visual symptoms on a 0 to 4 scale and (ii) quantitative measurement of acetic acid by subjecting the juice from three macerated berries to HPLC analysis (2). The ethanol content of each sample will be determined similarly.
- Understand the role of odors in mediating interactions between sour rot and Drosophila fruit flies.
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 30 replicates per day for three days for a total of 90 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 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.
Video recordings of fly behavior in response to inoculated berries
Observation arenas will consist of rectangular polystyrene Petri dishes, containing inoculated and non-inoculated berries as a treatment, separated by 5 cm. The arena will be illuminated by red light (λmax = 635 nm) from above and monitored using a webcam system. At the beginning of an experiment, a single female fly will be introduced into the arena and habituated to the new environment for five minutes. Each insect will be monitored for twenty minutes using a digital camera above the arena, recording to Etho Vision XT (Noldus Information Technology Ltd.). There will be six replications per day, and each set of the trial will be conducted for three days in laboratory conditions with a total of eighteen replications. The tracking software will be used to quantify the movement of insects throughout the trial within a predefined arena, measuring a) a number of times D. melanogaster lands on inoculated and non-inoculated berries; (b) time spent walking on inoculated and non-inoculated berries, and (c) time invested before the first attempt to oviposit d) time spent feeding by D. melanogaster on inoculated and non-inoculated berries.
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 loading ‘lme4’ package (3) in R software version 3.6.3 (4).
Volatile profile analysis and behavior study
The headspace volatiles collected from inoculated and incised non-inoculated berry samples will be analyzed using gas chromatography and mass spectrometry (GC–MS). Each experimental unit will consist of inoculated and non-inoculated but incised berries placed in separate cylindrical glass containers. The airflow will be generated by a vacuum pump with an air stream through the device from the air purifier cartridge to the trap cartridge. Five independent sets of replicates will be used for the volatile analysis per day, and the trial will be replicated three times over three weeks. Analysis of chromatograms obtained from the samples after analyzing in GC-MS will be overlaid, and the peaks will be identified using the NIST library. The internal standards will be used in an eluted solution as a reference to calculate the amount of each compound present in the treatments. Differences in volatile composition and abundance between treatments will be evaluated by comparing chromatograms. The amount of compounds in two treatments will be analyzed by univariate one-way ANOVA. The variance among the compounds for each treatment will be analyzed using multivariate analysis: Principal Component Analysis in R software version 3.6.3 (4). To assess the biological relevance of identified compounds or blends of compounds, electrophysiological responses of the antennal sensilla will be evaluated for the compounds using GC-EAD (gas chromatography coupled with electroantennographic detection). Additional behavioral bioassays of flies will be conducted in response to the behaviorally active compound or blend of compounds.
- Evaluate whether infestation by D. suzukii facilitates the subsequent D. melanogaster infestation
For laboratory bioassays, gnotobiotic flies will be produced by administering yeast and acetic acid bacteria to dechlorinated eggs of D. suzukii in combination (5), with slight modifications. Olfactory preference of D. melanogaster in response to the cues associated with berries with ovipositor probings of D. suzukii compared to artificially probed berries will be evaluated. The experimental unit will consist of a single female D. melanogaster, and treatments will consist of three fly-probed berries and three artificially probed berries. The movement of insects will be tracked, measuring a) the number of times D. melanogaster lands (b) time spent walking on inoculated and non-inoculated berries, and (c) time spent before the first attempt to oviposit on or near the probes d) time spent on feeding by D. melanogaster on fly probed and artificially probed berries. The hypothesis is that D. melanogaster females will increase time and activities on berries with previous exposure to D. suzukii oviposition compared to berries with mechanical injury.
A similar set of trials will be conducted to assess the preference of D. melanogaster when provided with berry plus D. suzukii larvae and incised berry as a control treatment. For both experiments, there will be three replicated trials per day. Each trial will be conducted for three days in the lab with a total of eighteen replications. The movement and behaviors of D. melanogaster will be tracked to assess the specific behaviors as mentioned previously on berries with and without D. suzukii larvae.
Similar experiments using berries containing probes/larvae of axenic flies (devoid of gut microbes) will be evaluated and compared with the berries containing probes/larvae of either axenic or gnotobiotic D. suzukii flies to understand the role microbes play in D. melanogaster behaviors.
The software will be used to track the behaviors of D. melanogaster in response to the probing, larval presence of D. suzukii. The experimental setup and time duration for both experiments will be similar to the open arena bioassay in objective 1.
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)
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 7days 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.
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. 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 (Figure. 1 in attached slide) Similarly, when we reared out flies from the bagged clusters subjected to injury and Drosophila fruit fly treatments in deli-cup in the laboratory, more adult flies emerged from the berries (Figure. 2 in an attached slide) 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 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. However, this first-year study has somewhat elucidated the role of multiple insects in sour rot etiology, providing new insights into sustainable disease management strategies. The second-year study will confirm our results and provide more insights into the role of injuries in sour rot disease etiology.
Education & Outreach Activities and Participation Summary
We will communicate our research results to growers, extension educators, and other relevant stakeholders through regional and statewide newsletter articles, presentations at winter grower meetings, summer field tours, and social media. We also will present results at scientific meetings (Entomological Society of America, Cornell Recent Advances in Viticulture and Enology or CRAVE) and publish articles in discipline peer-reviewed journals. Specifically, results will be developed into a grower-oriented research article for Appellation Cornell and included in the spring entomology update written by Ph.D. advisor Dr. Greg Loeb and published in regional grape extension newsletters (Finger Lakes Vineyard Update, Lake Erie Regional Grape Program Vineyard Notes, Long Island Fruit & Vegetable Update) as well as made available through Cornell Fruit Resources web page (fruit.cornell.edu). Results from this project will also be included in updates for the New York and Pennsylvania Pest Management Guidelines for Grapes. We also plan to produce one extension video for grape growers discussing the findings from this study made available on YouTube.
From our first-year trial to investigate third project objective, we learned that injuries are 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.
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 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 on 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 role as a vector in sour rot disease etiology in vineyards.
- ASSESSING ROLE OF INSECTS IN SOUR ROT DISEASE ETIOLOGY IN VINEYARDS (Conference/Presentation Material)