2016 Annual Report for GNE16-115
The role of insects as fire blight vectors: Implications for sustainable disease management in Northeast apple orchards
Fire blight (Erwinia amylovora) is a devastating bacterial disease of apple, causing severe damage and economic loss in the Northeast, where over 20% of the nation’s apples are produced. To date, growers depend on streptomycin applications to control the disease, but this method is not sustainable due to occurrences of, and continued risk for, antibiotic resistance. Though insects have long been implicated as fire blight vectors, the potential benefits of controlling vectors as part of a fire blight management program are unknown. The purpose of this project is to investigate interactions between fire blight and potential insect vectors to increase management sustainability.
Our first step is to identify major insect vectors in the field and confirm vector capability in the lab. Starting at bloom, we collected insects on yellow sticky cards in an experimentally infected orchard across 20 weeks. Pollinators were collected in vials during bloom and frozen until analyzed. Based on previous research, we focused on hemipterans (hoppers) and dipterans (flies) on sticky cards. Four hopper species were removed from the cards (potato leafhopper, speckled leafhopper, aster leafhopper and meadow spittlebug) and each individual was tested for fire blight. Of the four hoppers, speckled leafhopper produced the highest positive rate at 7%, while only ~1% of meadow spittlebug and 0% of aster leafhopper tested positive. Analysis for potato leafhopper is ongoing, but the projected positive rate is between 1% and 3%. Analysis of flies will begin in January 2017. Guts were extracted from pollinators and while analysis is ongoing, none have tested positive thus far. We will test bee bodies for fire blight starting January 2017.
Following vector confirmation, we will conduct molecular and behavioral assays to further understand the relationship between the pathogen and its vectors. We have colonies of potato leafhopper (Empoasca fabae) and Drosophila melanogaster to run acquisition, transmission, and behavioral assays. We will begin these experiments in January 2017.
Objective 1: Identify candidate insect vectors of E. amylovora.
Description: Existing data on insect transmission of fire blight is outdated and characterizes the vectoring process as passive. We hypothesize that certain insects actively vector fire blight by transporting the pathogen internally. We will conduct field surveys throughout the growing season to monitor changes in community structure, using molecular methods to confirm E. amylovora presence in insect guts. We will focus on pollinators visiting blossoms; hemipterans who provide entry wounds for shoot colonization by E. amylovora; and dipterans visiting oozing cankers on diseased trees. Finally, we will use live captured candidate vectors to conduct laboratory acquisition and transmissions assays to fully confirm vectoring capability. These data will be instrumental for monitoring and control of insect vectors in commercial and small holding orchards.
- First field season (May 2016-September 2016) completed. Each week, 20 trees across 4 blocks (5 trees per block) were selected at random and 2 yellow sticky cards were deployed at knee (trunk) and shoulder (canopy) level of each tree. Cards were removed after 1 week and new cards deployed on 20 new, randomly selected trees for 20 weeks. Total of 800 cards for the full field season were visually analyzed and abundant insects were identified for further processing.
- Roughly 1500 hoppers across four species identified, removed from yellow sticky cards, DNA extracted, and tested for fire blight via PCR/gel electrophoresis.
- Roughly 400 bees across three families collected over 2 weeks during bloom. Each insect was gut extracted, guts were DNA extracted and tested for fire blight via PCR/gel electrophoresis.
- 3 fly morphospecies identified on sticky cards for processing.
- Potato leafhopper colony established for laboratory acquisition and transmission assays. Acquisition and transmission assay designs tested for efficacy.
Objective 2: Investigate persistence of E. amylovora within insect guts.
Description: Two novel type III secretion systems (T3SSs) in the fire blight genome were recently characterized as non-pathogenic, showing greater genetic homology to human enteropathogens and insect endosymbionts rather than phytopathogens. The role of these secretion systems in the fire blight disease cycle is unknown. We hypothesize that these T3SSs mediate interactions between vectors and E. amylovora, allowing the pathogen to persist in the insect gut. We will challenge insects with ΔT3SS mutant or wild type (WT) E. amylovora and use qPCR at multiple time points to evaluate pathogen abundance. We will compare insect acquisition and transmission of ΔT3SS and WT E. amylovora in laboratory assays. These data will provide insight into the cellular relationship between fire blight and its vectors, with implications for disease control in other agricultural pathosystems.
Accomplishments: We have not begun work on objective 2 with plans to begin experiments in fall 2017.
Objective 3: Evaluate insect preference for healthy or diseased apple shoots/blossoms.
Description: Many plant pathogens alter volatile profiles of their hosts, preferentially attracting vectors. In apple, changes in shoot and blossom volatile profiles due to fire blight infection are unknown, as is the resultant impact on insects. We hypothesize that infection induces changes in volatile emissions from blossoms and shoots, resulting in preferential insect aggregation and visitation on diseased tissue. We will conduct choice and non-choice bioassays to evaluate insect preferences for diseased tissue. We will also characterize the volatile profiles of diseased and healthy shoots and blossoms to identify key chemical differences between the two. These data lend insight into multi-partite interactions between plant pathogens, their hosts, and insect vectors, justifying future assays aimed at identifying and utilizing behavior altering compounds for vector management.
Accomplishments: We have not begun work on objective 3 with plans to begin experiments in early 2018.
Starting in the first week of May 2016, we deployed 40 yellow sticky cards into an orchard experimentally infected with fire blight. Cards were replaced on a weekly basis and trees were selected at random each week for 20 weeks. Cards were stored at -20℃ until processing, which was usually done during the week they removed from the orchard. As stated above, four hopper species were abundant across the season and these insects were removed from the cards, cataloged, and stored at -20℃ until they were DNA extracted. In total, we removed over 1500 hoppers from sticky cards and tested them for fire blight. We have completed analysis on three out of four of these hopper species, with the data presented in the summary. We still have to complete analyses on potato leafhopper, which will be complete by the end of January 17. We have identified flies on the sticky cards that we will process, but have yet to begin removing those insects from the cards. We were surprised to see such a high positive rate in the speckled leafhopper (Paraphlepsius spp.) as these insects had not previously been implicated as pests of apple. This finding has distinct potential significance, as this insect could be a target for management.
Pollinators were sampled separately on a daily basis from bloom to petal fall. For pollinators, five trees were selected at random and observed for 10 minutes. Any insect landing on flowers were captured in sterile vials and frozen at -80℃ until processing. Bees were either identified on sight or in the lab on the same day of their capture. In total, almost four hundred bees were captured across the two-week bloom period. Almost 50% of the bees were Lasioglossum pilosum, a small solitary bee, another ~18% were Andrena miserabilis, a small mining bee, and another ~15% were honey bees (Apis mellifera). The final ~17% of bees were miscellaneous captures across various families and genera.
Bees were stored until late September 2016, at which point sets of 12 bees were gut dissected and guts were DNA extracted. For bees that were too small for gut extraction, abdomens were removed, the external surface was washed, and entire abdomens were DNA extracted. Bee guts were tested for fire blight via PCR and gel electrophoresis. This work concluded in mid-December 2016, and while none of the gut extractions tested positive for fire blight, the rest of the insect has not yet been tested. In January 2017, I will begin DNA extractions on the bee bodies and test those for fire blight as well. We expected to get a fair amount of positives in the bee guts, and we are not sure why 0% of all bee guts tested positive. This result is interesting and we will use our second field season in 2017 to try and corroborate these data before making broader inferences.
The above paragraphs describe the progress made on objective 1. We have made significant progress, but the sheer number of samples (~2000 with more to go) was unexpected. The higher number of samples delayed our timeline on objectives 2 and 3, bumping back the projected starting date for objective 2 to after the 2017 field season. Our goal is to complete objective 1 by the end of 2017. The only components remaining here are the 2nd field season, which we will use to corroborate data generated in the first field season, and the laboratory acquisition and transmission assays, which we are planning to start January 2017. Some of the assays for this project, such as the DNA and PCR protocols, are new to the lab, so they took some optimizing before we could effectively process samples. The acquisition and transmission experiments will also be new procedures, so they will likely take some optimizing that was not previously accounted for in the timeline. At this point the overall plan of work has not changed, but we are bumping back the start dates of objectives 2 and 3 to make sure we complete objective 1 properly.
Impacts and Contributions/Outcomes
This project is still in its preliminary stages, so the impacts and sustainability contributions are minimal at this point. The identification of a new potential fire blight vector (speckled leafhopper) may have long term management implications and our current findings will be important for monitoring vectors. We envision a fire blight management program that integrates vector management, which requires tracking the most capable vectors and advising growers on control strategies during peak vector prevalence. Our major focus right now is publicizing this research to growers. In July 2016, I gave a presentation about our work and our goals to over 300 apple growers from around the world at the Cornell Fruit Field Day. I’ve also given an update on preliminary data to a panel of apple growers from across New York State and am scheduled to give a talk in late January 2017 at the Cornell Entomology Symposium. An article on this research was also published in Good Fruit Grower in September 2016. Our hope is to use these communication channels to disseminate our findings to the Northeast USA and beyond.
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