Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

The goal of this procedure is to describe a push-pull method for collecting blueberry volatiles and to investigate the response of undamaged blueberry branches to volatiles omitted from herbivore damaged branches with implants. This is accomplished by first collecting local and systemic volatiles from blueberry plants damaged by gypsy moth, caterpillars mechanical damage, and by an exogenous application with the phyto hormone mytho Jasmine eight. The second step is to expose undamaged blueberry branches to volatiles from branches damaged by the herbivore gypsy moth, and to then collect volatiles from these exposed branches before and after herbivore feeding.

The third step of the procedure is to extract the collected volatiles. The final step is to inject the extractive volatiles onto a gas chromatograph. Ultimately, results can be obtained that show induction of blueberry volatiles by herbivore feeding and their role in intra plant signaling through a push-pull, volatile collection system and gas chromatography.

The main advantage of the push-pull system over other existing techniques, such as the solid phase micro extraction or pmi, is that the collected volatile samples are absorbed at volumes that allow for multiple analysis. This method can help answer questions in the field of insect plant interactions, such as the role that plant volatiles play in plant defenses, and also on pollination To induce volatiles by herbivore damage. First bag two branches of blue brew plants with a spun polyester sleeve.

Then place three gypsy moth caterpillars in each bag and allow them to feed on the plant for two days. Meanwhile, the control plant does not receive any caterpillars. On day three, collect the volatile emissions by sampling the plants in the greenhouse using a push-pull system.

Place the above ground portion of the plant, including the caterpillars in the herbivore damaged treatment inside a four liter volatile collection chamber. Support the base of the plant by a two piece guillotine, then collect the volatiles in filter traps filled with 30 milligrams of Alltech, super cute absorbent by evacuating the air from the chambers at a rate of one liter per minute. The purified air enters the top of each chamber at a rate of two liters per minute to induce volatiles by mechanical damage.

Punch two seven millimeter holes at the base and up a portion of each of the five leaves per plant at the end of days one and two in order to mimic the amount of leaf area removed by gypsy moths. Similarly, on day three, collect the volatile emissions with the same method as before to induce volatiles by methyl Jain.Eight. Treat blueberry plants at 4:00 PM with 10 milliliters of either one or 1.5 millimolar methyl Jain eight in a 0.1 tween 20 solution using a spray bottle.

Meanwhile, spray the control plant with 10 milliliters of a 0.1%tween 20 solution without methyl Jain eight. Next, keep the plants inside a 17 centimeter diameter and 35 centimeter high plexiglass chamber in the greenhouse for 15 hours. Then collect the volatile emissions to induce volatiles systemically.

Bag a lower branch of the blueberry plant with a spun polyester sleeve. Then similarly to the local volatiles induction by herbivore damage placed six gypsy moth caterpillars in the bag. The damaged branch remains outside the volatile collection chamber.

While the branches above the damaged branch are placed inside the chamber. Allow the caterpillars to feed on the bottom branch for two days, starting on day three. Collect volatiles from the undamaged portion of the damaged plant for seven consecutive days.

Then treat the control plant in a similar manner except for the placing of caterpillars to determine the degree of vascular connectivity among different branches within a blueberry plant. Cut the end portion of a lower branch of the plant to support and allow absorption of the floral water. Pick containing six milliliters of Rodin B dye.

Then monitor the movement of the dye through the plant daily for seven days. After seven days. Visually assess the amount of red staining from different positions of the plant to expose the branches to h IPVs bag, one lower branch with a polyester sleeve and place six jui moth caterpillars in the bag.

Cage the plant inside a plexiglass chamber. Allow the insects to feed on the plant for two days, such that adjacent branches are exposed to volatiles emitted from the induced branch. On day three, place the exposed branches inside the volatile collection chamber, leaving the insect injured branch outside, collect volatiles from HIPV exposed branches for 24 hours.

Then measure the leaf area of the lower branch consumed using Scion image software to determine whether the plant is primed. After HIPV exposure, expose half of the plants to HI PVS from the induced adjacent branches and half of them to the undamaged branches as described above. On day three, place the plants in the collection chamber for 24 hour collection of volatile emissions and then place four gypsy moth caterpillars on each plant.

Then measure the damage leaf area. Afterwards, elute the collected volatiles from the super Q traps with 150 microliters of DIAM methane, using 400 nanograms of en octane as an internal standard. Separate and quantify the compound by a gas chromatograph, which is equipped with a flame ionization detector and an ENT HP one column and uses helium as carrier gas.

Following these procedures, the identification of compounds was carried out on ovarian 3, 400 gas chromatograph coupled to a Finnegan mat 82 30 mass spectrometer. The GCMS was equipped with a selco MDN five S column and used helium as the carrier gas. The mass spectrometer was operated in electron ionization and the total ion chromatogram mode at 250 degrees Celsius to tentatively identify the compounds.

The spectral data were compared with those obtained from the NIST library and the gas chromatograph retention index database. In addition to verify identification, their retention times were compared with those of the commercially available compounds. Shown here are the typical chromatographs of undamaged blueberry leaves and leaves damaged by gypsy moth.

Caterpillars volatiles remitted at very low amounts from the undamaged blueberry leaves. However, when leaves are damaged by gypsy moth, caterpillars emission of volatiles increased dramatically. The mechanical damage and the caterpillar damage both increased local volatiles emissions compared to the control.

This figure shows that methylate treatment also increased volatiles emissions from blueberry leaves. However, no evidence of systemic induction of volatiles from undamaged leaves of gypsy moth damaged plants was found seven days after the initial feeding damage, which indicated that there was no internal signaling. In addition, after one week, very slow movement of the red dye was observed among branches of blueberry plants.

This graph shows the amount of volatiles admitted from branches that were exposed to HI PVS and unexposed branches. HI PVS did not seem to affect volatile emissions in neighboring undamaged blueberry branches. This figure shows the amount of feeding by gypsy moth caterpillars on blueberry branches exposed to HIP VS and unexposed branches.

Caterpillars on HIPV exposed branches consumed less foliage compared to those on unexposed branches. Furthermore, amounts of volatiles admitted per amount of leaf area consumed in HIPV exposed branches were fourfold higher compared to unexposed branches, indicating that the leaves from HIPV exposed branches were more responsive to herbivore. This technique paved the way for researchers in the field of insect chemical ecology to explore trade tropic level interactions.

These are interactions among plants, herbivores, and naturales in agricultural crops. After watching this video, you'll have a good understanding on how to collect, extract, separate and analyze volatiles emitted from plants.