Chestnut Inoculation Procedure

Inoculation Procedure - click here to go to information on how to inoculate chestnut trees
Selection Procedure - click here to go to information on how we select for resistance and American type in our orchards after inoculation.

The process of inoculation is highly dependent on weather. Keep your eye on this website for weather updates. If rain is forecast for an entire scheduled inoculation day, inoculations will be held the following day (see rain date); however, if it looks like the rain will be spotty, we'll wait until passing showers finish their business. Should the forecast call for extended periods of cold and rain following the inoculation day, likewise, inoculations will have to be postponed. Keep your fingers crossed for fair weather.

Inoculation is the process by which we judge the resistance of a chestnut tree. During the inoculation procedure, we take a plug of chestnut blight and insert it directly into the trunk of a chestnut tree.

The following approach is used for larger chestnut trees. In general, we use these procedures in our backcross orchards, where the backcross trees will exhibit no-to-moderate resistance.

Before You Inoculate:

1. Tree shelters must be removed summer before to allow trunks to harden to a natural condition.
2. Diameter at breast height or at top of tree shelter must be a minimum of 1 1/8" - larger
diameter is preferred.
3. Tree must be in its fifth year of growth.
4. Mark trees to be inoculated in advance unless you experienced as an inoculator.
5. Early June is generally the time to inoculate.

Inoculate your orchard when the majority of the trees averate 1 1/8" DBH or higher. We sometimes recommend waiting until the average DBH approaches 1 1/2" DBH. Attempt to inoculate all trees in the same orchard at the same time in order to be able to select from an entire backcross line at once.

Supplies for 150 trees:

Inoculum - order from Fred at Meadowview Research Farms
1 quart 190-proof grain alcohol (Everclear or Vodka)
1 pint 70% ethanol alcohol

--- Why do we use two different types of alcohol??

>We use the 95% for flaming tools; it's more flammable than 70%.

We use the 70% for surface disinfection (or disinfestation, more properly) of bark surface. The 70% is much more lethal on contact to living organisms than other strengths, because it readily penetrates cell walls and plasma membranes; the organisms get "wetter" faster in 70%, I guess because it has the same polarity as the cell walls and plasma membranes --this ties into the idea of how it can be difficult initially to get something wet. For instance, if you spill water on your clothes, you often can wipe off a significant fraction of the water before it soaks in. For living organisms, the 70% EtOH would soak in fastest, in comparison to 50% or 90%; this might also be involved with the 70% is still highly lethal to tissues, whereas 25% might not be. I'm not sure about 50%. The idea is to kill the algae, etc, on the surface of the bark, so they don't interfere with growth of the inoculum from the agar disk into the surrounding tissue.

10 rolls ½"-width masking tape
1 cork borer sharpener
4 caddies to carry supplies - or, a 5 qt. ice cream bucket with handle works well
6 rags per team
6 cigarette lighters per team
3 cork borers # 0 or # 1 size (about 0.4cm diameter, or 3/16") per team
4 screw top test tubes for 190-proof alcohol per team
3 small bottles to carry extra alcohol
3 spatulas per team - the spatulas are about 8 " with a 1" long sharp end on one end and with a 1" long blunt end on the other.
Couple of pint or half pint jars for ethanol weight reduction

Like most of the procedures in this business, the process of inoculation is highly dependent on weather. Keep your eye on this website for weather updates. If rain is forecast for an entire scheduled inoculation day, inoculations will be held the following day (see rain date); however, if it looks like the rain will be spotty, we'll wait until passing showers finish their business. Should the forecast call for extended periods of cold and rain following the inoculation day, likewise, inoculations will have to be postponed. Keep your fingers crossed for fair weather.

1. Organize team of four to six - one plugger, one to two inoculaters, one to two tapers, and a recorder.
Prepare supplies for each team member.

Plugger - sanitizes cork bores by flaming with 190-proof grain alcohol
(Warning - don’t stick bore barrel into alcohol too deeply or you will end
up with a blow torch)(Beware - you can’t see the blue flame, but you can feel it); prepares wipe rag with 70% ethanol alcohol to sanitize areas of trunk to be inoculated, and prepares caddy with test tube secured and filled with 190-proof alcohol, extra alcohols, 2 lighters and rags.

Inoculators - each prepares caddy for alcohol bottle, test tube securely taped to caddy, 2 cigarette lighters, spatula, 2 petri dishes of inoculum well identified with inoculum name, a rag. Mark caddy also with the strain of inoculum being used. Use sterilized cork bore to cut plugs of mycelium. Be sure to avoid making plugs of jell with no mycelium present. Avoid the center of the culture which may be contaminated with excretions from and dying cells of mycelium.
Tapers need ½" masking tape and good attention span. Recorder needs list of trees by number to record trees which are inoculated, number of holes inoculated, and any errors made in the process. Date, location, orchard name, list of team members should be added.

2. Plugger selects qualifying tree and four inoculation sites on main trunk. Avoid blighted areas, crotches, and green stripes which lay down the bark of the tree, known as rhytidome initiators (about rhytidome). Holes should be placed about 12"-18" apart. Wipe sites with alcohol rag. Sanitize cork borer and push and twist wrist a half turn til you hit hardwood. You can accumulate plugs for some time before emptying and flaming. If you can’t find four suitable sites, you may use just two, but most trees should be large enough to select 4 sites. Don’t get too far ahead of inoculators or the holes will dry out.

3. First inoculator uses less virulent strain (SG-1) of blight in two upper holes. Locate all four holes first. Fire both ends of the spatula, then slip one plug of jell onto first end and apply it to the top hole, mycelium in toward the cambium. Pat plug gently in. Wipe off end of spatula on pants or a cloth. Use other end to pick up second plug and apply as above in second hole from the top. If you put the mycelium in the wrong hole, mark the hole with permanent magic marker with the (SG-1) in several places around the hole and have the recorder make note of it. Go on to next tree. Fire your spatula for each tree. Keep petri dishes shaded so mycelium will not dry out and die. You may carry two dishes, but replenish supply from central source rather than trying to carry it all with you.

4. Second inoculator uses the same process using the more virulent strain (155) of blight on the two lower holes.

5. Tapers are the control people. Make sure the proper hole is inoculated with the proper mycelium. Immediately after your inoculator fills the hole, cover it with masking tape by circling the entire trunk or limb with the tape, doubling the tape over the hole. This keeps the mycelium from drying out. Make sure no trees that have been bored are missed. One taper follows the same inoculator all the way through. Quality control!