PLANT, MICROBES AND ODORANTS INVOLVED IN INSECT HOST PLANT LOCATION: WHOS MAKING THE MESSAGE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004404
• Project Start Date: Oct 23, 2014
• Project End Date: May 31, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456

Performing Department
Geneva - Entomology

Non Technical Summary
Plant eating insects are important pests of agricultural crops in NY State. Understanding the role of odors in their host plant seeking behaviors allows for development of new tools using semiochemicals to affect insect behavior and control population levels. This research also provides a better understanding of how host preference can evolve and insects adapt to new hosts. Significant progress has been made in our ability to analyze odors from plants that affect insect behavior and a number of studies have demonstrated the importance of odors in insect foraging behavior. Use of these chemicals in environmentally safe management programs constitutes an exciting new research area.However, it also is clear that the origin of the molecules in the plant odor space is complicated by the fact that in addition to plant-produced odors, other key odors might be produced by microbes (fungi and bacteria) associated with the host plant. This adds an important extra dimension to the concept of a plant's 'odor space' that must be considered in the analysis of host odors involved in plant-consuming insect foraging behavior. This information will provide exciting new possibilities for applied chemical ecology by incorporating organisms that are able to produce odors that adversely affect the behavior of a key pest.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	1139	1130	50%
211	1110	1130	50%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1110 - Apple; 1139 - Grapes, general/other;

Field Of Science
1130 - Entomology and acarology;

Keywords

semiochemicals

host preference

flight tunnel

microbes

fungi

bacteria

grape berry moth

Goals / Objectives
The goal of the project is to determine to what extent the volatile profile from a host plant or fruit is composed of volatiles from the plant vs volatiles being produced by microbial agents on the plant material.

Project Methods
Objective I. Collect headspace odors from treated (surface sterilized) and untreated grape plants in the shoot and fruiting stages. We will use Vitis riparia, to characterize volatile profiles from microbe dwelling and clean plants. We will use shoots, or bunches of grape berries, V. vinifera, red flame variety or Red Delicious apples for R. pomonella, fruit from Downy red hawthorn, Crataegus mollis.Shoots and grape berries, apples, or hawthorn fruit will be surface-sterilized by wiping surfaces with 70% ethanol, then dipping for 5 seconds in 70% ethanol, followed by a second wipe, and then washed twice with sterile distilled water for 1 min/wash. Treated material will then 1) be placed on LB Broth (10g SELECT Peptone 140, 5g SELECT Yeast Extract, 5g sodium chloride) or potato dextrose agar (PDA; Difco Laboratories, Sparks, MD), or 2) taken within 5 min. for volatile collection or flight tunnel assay.Objective II. Identify odors detected by female moths. We use a push-pull system to collect the headspace volatiles of live sterilized and untreated plant material. The system has a custom-made bell-shaped glass chamber with two air-in adapters on the top and four air-out adapters at the bottom wall. Filtered clean air is pushed into the chamber and volatiles from the headspace are drawn by a vacuum pump onto four activated charcoal filters at 0.5 l/min/filter. The volatiles are eluted with 1 ml hexane every 24 hours and then combined.Coupled Gas Chromatographic-Electroantennographic Detection (GC-EAD) Analysis. Coupled GC-EAD analysis is performed using a Hewlett Packard 5890 Series II gas chromatograph equipped with a nonpolar EC-1 capillary column or a polar EC-Wax Econo-Cap column in the splitless mode. The oven temperature is programmed at 40ºC for 5 min, then increased by 15ºC/min until 250ºC, and held for 5 min. Injector and detector temperature are set at 280ºC and 270ºC respectively. Nitrogen gas is the carrier gas with a flow rate of 2 ml/min. Septum purge flow rate is set at 3 ml/min and the split vent flow rate at 55 ml/min. The column effluent is split by a ratio of 1:1 in the oven via a "Y" glass splitter and is led simultaneously to the flame ionization detector (270ºC) and to the heated EAD port (270 ºC).moths are reared in walk-in environmental chambers at 26ºC and 60% RH under a 16:8 (L:D) photoperiod. Adults are fed with 50% honey and water soaked on cotton. Adults mate in rearing cages and oviposit on V. vinifera seedless grapes. A whole head is removed from a 2-day-old virgin female and both antennae are positioned between two gold wire electrodes, which were immersed in two saline-filled micropipettes in a small acrylic holder. . The antennal holder is placed inside a humidified cooling condenser. A minimum of 10-15 different antennal pairs (2-3 runs/pair) is used for analysis.Chemical Identifications. GC-mass spectrometry (GC-MS) is carried out with an Agilent 6890 GC coupled with a 5973n mass spectrometer equipped with a nonpolar DB-1 capillary columnor a polar EC-Wax Econo-Cap capillary column. Helium is the carrier gas at an initial head pressure of 54 kPa at constant flow rate (1.0 ml/min). Injector and interface temperature are set at 280ºC and 260ºC, respectively. The oven temperature is programmed at 40ºC for 5 min, then increased by 15ºC/min to 250ºC, and held for 5 min. Volatile compounds are identified by mass spectral matches to library spectra as well as by retention time matches to available known standards. The EAD active compounds are verified by the GC-EAD analysis with authentic standards.Objective III. Determine behavioral activity of extracts and synthetic odor blends in the flight tunnel. Surface-sterilized or untreated shoots/fruit, as well as adsorbent extracts, will be tested with mated female GBM for behavioral activity in the flight tunnel. The flight tunnel is 2 m L by 0.6 m W and 0.6 m H with wind speed is set at 0.25 m/sec at the point of release of moths. Light is provided from above using eight 25 W incandescent bulbs, set to 25 lux to mimic dusk conditions. Temperature and relative humidity are in the range of 23-25ºC and 50-60%.Freshly cut shoots for both treatments are placed in water picks just before placement at the upwind end of the flight tunnel. Fruit are placed on a metal stand at the same location as shoot bouquets. Adsorbent extracts, prepared in hexane at concentrations designed to provide desired doses when applied in a known volume are applied to red rubber septa and placed in a copper tube holder in the same position as the plant shoots or fruit at the upwind end of the tunnel.Mated 4-day-old females are tested individually around dusk (a 2 hr period including 1 hr before and after the end of the light schedule in the environmental chamber), recording behavior for 5 min per moth. Each moth is scored for making an upwind-directed flight in the odor plume or landing on the target.

Progress 10/23/14 to 05/31/18

Outputs
Target Audience:Target audiance inludes academic institutions, department seminars, outreach education events for the public. Results will contribute to development of plant based olfactory cues in pest management schemes. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the course of this project three summer research opportunities were provided forundergraduate students, Sara Volo, Yuxi Liu, and Jonathan Thrall, at Hobart and William Smith Colleges, Geneva NY. The students werementored by myself and graduate student Michael Wolfin. A Senior Technician, Steve Hesler, also participated by helping to maintain the plants in a greenhouse, and another technician, Shin-Young Park, maintained the moth colony. How have the results been disseminated to communities of interest?For each summer of undergraduate participation a poster was prepared and presented by the students at the Fall Science Schlars symposium at Hobart and William Smith colleges, Geneva. PI Linn also presented results of these studies at the International Congress of Entomology in Orlando Florida. What do you plan to do during the next reporting period to accomplish the goals?This is the final report. The project ened in March 2018.

Impacts
What was accomplished under these goals? The goal of the project is to determine to what extent the volatile profile from a host plant or fruit is composed of volatiles from the plant vs volatiles being produced by microbial agents on the plant material. We successfully established a protocol for sterilizing plant tissue using an ethanol dip and wipe procedure. After dehydration the plant material was used for volatile collections, flight tunnel tests, and plating on PDA. We found that 1) sterilized plant tissue released the exact same volatile profile as unsterilized plants, 2) female grape berry moths were attracted to sterilized plants in the same proportion as unsterilized plants, and 3) there was minimal fungal growth on the agar plats after 48 hours. Our conclusion is that the previously identified and behaviorally active volatile blend from grape is a product of the plant and not surface microbial agents. In 2017 we analyzed, using GC-mass spectrometry that volatile collections from grape plants in the field over the course of the summer have more complicated volatile profiles than our plants maintained in the greenhouse. These volatiles were shown to be the result of 1) microbial agents growing on the leaf surfaces, evidenced by collections made from sterilized plants from nature, and 2) from metabolism in the plant as a result of insect damage, principally from Japanese beetles. However, preliminary flight tunnel tests indicate no adverse effects on grape berry moth flight behavior and so we are still working under the conclusion that the behaviorally active blend of volatiles for moth orientation is not a product of microbial agents.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Wolfin, M. S., Volo, S. L.*, Chilson III, R. R.*, Liu, Y.*, Cha, D. H., Cox, K., Loeb, G. M., Linn, C. E. Plants, microbes, and odorants involved in host plant location by a specialist moth: Who⿿s making the message? Entomologia Experimentalis et Applicata.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:General public in terms of food production Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the summer of 2017 this project provided summer research opportunities to an undergraduate student, Jonathan Thrall, at Hobart and William Smith Colleges, Geneva NY. Jonathan was mentored by myself and graduate student Michael Wolfin. A Senior Technician, Steve Hesler, also participated by helping to maintain the plants in a greenhouse, and another technician, Shin-Young Park, maintained the moth colony. How have the results been disseminated to communities of interest?The material was presented Jonathan Thrall as a poster presentation at the Fall summer scholars symposium, Hobart and William Smith Colleges. The material also constitutes one of Mike Wolfin's dissertation chapters, completed in November 2017. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the investigation of moth responses to volatile collections from grape plants in the natural environment.

Impacts
What was accomplished under these goals? We successfully established a protocol for sterilizing plant tissue using an ethanol dip and wipe procedure. After dehydration the plant material was used for volatile collections, flight tunnel tests, and plating on PDA. We found that 1) sterilized plant tissue released the exact same volatile profile as unsterilized plants, 2) female grape berry moths were attracted to sterilized plants in the same proportion as unsterilized plants, and 3) there was minimal fungal growth on the agar plats after 48 hours. Our conclusion is that the previously identified and behaviorally active volatile blend from grape is a product of the plant and not surface microbial agents. In 2017 we analyzed, using GC-mass spectrometry that volatile collections from grape plants in the field over the course of the summer have more complicated volatile profiles than our plants maintained in the greenhouse. These volatiles were shown to be the result of 1) microbial agents growing on the leaf surfaces, evidenced by collections made from sterilized plants from nature, and 2) from metabolism in the plant as a result of insect damage, principally from Japanese beetles. However, preliminary flight tunnel tests indicate no adverse effects on grape berry moth flight behavior and so we are still working under the conclusion that the behaviorally active blend of volatiles for moth orientation is not a product of microbial agents.

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:General public in terms of food protection Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided summer research opportunities to an undergraduate student, Yuxi Liu, at Hobert and William Smith Colleges, Geneva NY, and a high school student, Ronald Chilson, Penn Yan Academy. They were both mentored by myself and graduate student Michael Wolfin. A Senior Technician, Steve Hesler, also perticipated by helping to maintain the plants in a greenhouse. How have the results been disseminated to communities of interest?The material was presented my Charles Linn at the 2016 International Congress of Entomology in Orlamndo FL, and asa poster presentation by Yuxi Liu at the Fall summer scholars symposium, Hobart and William Smith Colleges. What do you plan to do during the next reporting period to accomplish the goals?We plan to investigate the possible role of endophytic microbial agents in plant volatile production, and to determine at what point that a damaged plant begins to produce enough microbe metaoliosm to affect the volatile profiles.

Impacts
What was accomplished under these goals? We successfully established a protocol for steralizing plant tissue using an ethanol dip and wipe procedure. After dehydration the plant material was used for volatile collections, flight tunnel tests, and plating on PDA. We found that 1) steralized plant tissue released the exact same volatile profile as unsteralized plants, 2) female grape bery moths were attracted to steralized plants in the same proportion as unsteralized plants, and 3) there was minimal fungal growth on the agar plats after 48 hours. Our conclusion is that the previously idntified and behaviorally active volatile blend from grape is a product of the pplant and not surface microbial agents.

Publications

Progress 10/23/14 to 09/30/15

Outputs
Target Audience:General public in terms of food protection. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The studies will constitute one chanpter in Mike Wolfins doctoral dissertation. An undergraduate, Sara Volo, from Hobart and William Smith Colleges produced a poster presentatiuon of her project for the summer of 2015 and presented at the Fall Science Scholars symposium at the colleges. How have the results been disseminated to communities of interest?The results were presented in a poster at theFall Science Scholars symposium at Hobart and William Smith Colleges. What do you plan to do during the next reporting period to accomplish the goals?We lan to test other steralization protocols and to tes plants that have various stages of damage and thus possibly greater amounts of microbial presence.

Impacts
What was accomplished under these goals? We successfully designed a protocol for steralizing the surface of plant material that is attractive to female grape berry moths. The procedure involved an ethanol wipe and dip followed by rehydration with autoclaved water. Steralized plant material, when plated on PDA produced no microbe colonies after 96 hours, as opposed to control plants. Further, steralized plants were as attractive to femal moths as the control non-steralized plants. Finally gas chromatographic traces from headspace collections showed that steralized and non-steralized plants rpoduced identical traces.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Plants, microbes and plant volatiles involved in host plant selection: Who's making the message.