Integrated Practices

We attempted to integrate a mountain with the laboratory through the following activities.

1) Discusssion with Forestry Associations
2) Forest survey

Purposes

Know the current condition of pine-wilt disease in Kyoto city
Know what kinds of measures are taken specifically
Gain valuable opinions of people fighting against pine-wilt disease
Develop the design of our project
Obtain wild B. xylophilus

Results

We were able to make our project more relevant for the actual situation.
It was a big motivation for the project to personally confirm that the pine trees in Kyoto City are in a state of devastation.
We tried our RNAi feeding device, ‘B. x. Busters’ with wild nematodes.

Methods

Go to forestry associations managing the pine forest in Kyoto and listen to them.
Examine how much pine-wilt disease has spread by performing field work in the mountains.
Collect the branches and bark of pines affected by pine-wilt disease, and the soil around them to extract wild nematodes back in the lab.

Discussion with Forestry Associations

We learned through the internet that the pine-wilt disease has spread in Japan and thought we could solve this problem using genetic engineering. So, to understand the situation in Kyoto city where we live – how much pine-wilt disease has currently spread, what kinds of countermeasures are being taken against it, and what kind of device could be beneficial – we visited two forestry associations that were managing pine forests in Kyoto city and listened to them.

Figure 1 We had a discussion with staffs of Kyoto city Forestry Association.

Figure 2 We also had a discussion with staffs of Kyoto Osaka Forestry Association.

Through these discussions we were able to learn the following.

1. Drug spraying (as was posted on the Internet) is no longer used.
2. They are injecting pine trees every year to help the remaining pines, but the records have been decreasing year by year due to the expansion of the damage of pine-wilt disease.
3. Current measures against pine-wilt disease are accompanied by a physical burden on the foresters.
4. They want to use B. x. Busters if it is a light and easy-to-use.

Given the results of discussion, we chose to use modified yeast like a biological pesticide and decided to make yeast expressing dsRNA which will knock down some essential genes of B. xylophilus. By injecting this yeast into pine, yeast would settle inside the pine which we hoped would provide long-term protection from pine-wilt disease caused by B. xylophilus. (click here to see the mechanism)

Using a biological pesticide based on yeast, the currently required liquid measure of injected pesticides (300ml) could be greatly reduced. Moreover, once injected, it may not be necessary to inject into the tree for a long time. In that way it is expected that B. x. Busters will greatly reduce the physical burden of tree managers compared to the trunk injection that needs to be done every year.

Forest survey

Although we were stimulated by interviewing forestry staff about the current state of pine-wilt disease, we wanted to get more personally engaged and actually carried out a first-hand field survey of pine-wilt disease and collection of B. xylophilus in the back forest of Kiyomizu temple (national forest of Kodaiji mountain), which is considered to be a particularly damaged region in Kyoto City.

It is said that this mountain was once a forest mainly of red pines, but because pines were lost due to pine-wilt disease, it now became a forest of chinquapins. Surprisingly, we could only find 5 pines in about two hours of surveying (out of them, 2 pine trees seemed to already have pine-wilt disease).

Figure 3A We conducted a forest survey.You had better wear a long-sleeved shirt and long pants when you go to a mountain. B This is a sign of Kodaiji mountain. It says the forest type was gradually shifted to the evergreen broadleaved forest mainly of chinquapins after Meiji era (1868~1912).

From these experiences, we gained a new appreciation for the seriousness of the damage caused by pine-wilt disease, which became a great motivation to advance our project.

We also collected wood scraps from those 5 pines to extract wild B. xylophilus. Then, we were able to obtain 4 nematodes that were identified morphologically as B. xylophilus

Figure 4A We collected sawdust from withered pines to get wild B. xylophilus. B This is the way of extracting nematodes C We could get 4 wild B. xylophilus.

We attempted to cultivate them in order to test B. x. Busters with a wild B. xylophilus strain, but unfortunately we were unable to maintain them in the lab.

So we went to Yoshida mountain near Kyoto University and conducted a second and third forest survey and sampling of B. xylophilus. Yoshida mountain was once a place where many red pines grew like Kodaiji mountain, but here too, many pines withered by B. xylophilus, and now it was covered mainly by chinquapins. Moreover, there was a visible mark from trunk injection in all of the remaining pines; again we recognized the magnitude of the damage of pine wilt disease by B. xylophilus.

Figure 5A We found a lot of marks from trunk injection B A pine withered by pine-wilt disease.

We collected branches and bark from pines which were thought to be affected by pine-wilt disease and surrounding soils of them and extracted nematodes from those samples.

As a result, we were able to obtain many nematodes, and found many of the obtained nematodes were bacterial feeding nematodes by observing them through a microscope. We considered that B. x. Busters would need to minimize the influence on the environment, in particular the influence on the bacterial feeding nematodes, which would benefit the environment rather than the plant parasitic nematodes like B. xylophilus. (See the description for the difference between the two)

Figure 6 We could get a lot of nematodes.

Therefore, it is necessary to generate feeding RNAi using an essential gene sequence specific only to B. xylophilus and no other nematodes. We compared the sequences of essential genes of C. elegans, a representative of bacterial feeding nematodes, with those of B. xylophilus by BLAST. For example, this part of the AK2 gene which is a family of AK1 does not exist in C. elegans, and is therefore considered to be a candidate target sequence. We will continue searching for such sequences and evaluate dsRNA capabilities using B. x. Busters.

We also put both all the nematodes we collected along with the eGFP yeast we produced in the same plate for 1 day and observed them with a fluorescence microscope. Unexpectedly enough, it was found that nematodes other than B. xylophilus rarely eat yeast. And observing them carefully, diploid yeast is too big for their mouth to take in.

Figure 7 Unlike B. xylophilus, no fluorescence was detected. Scale bar : 100μm

From this, we concluded that B. x. Busters may act as a very specific pesticide capable of targeting only B. xylophilus through two strict parameters: the ability to feed on yeast, and the specificity of dsRNA.

Through this forest survey, we concluded the following:

1. Pine trees in Kyoto city are almost annihilated.
2. Many nematodes inhabit in and around pines in addition to B. xylophilus.
3. The decrease in pines greatly changes the forest ecosystem.

We could not collect many B. xylophilus from our three forest surveys. The following hypotheses may explain this:

1. The absolute amount of pines is so small that B. xylophilus no longer able to multiply.
2. Since withered pine trees are cut down every year, numbers of Pinus thunbergii Damnellomonidae (Japanese long horn beetle) drastically decreased, and therefore B. xylophilus are unable to move to healthy new pines.
3. The remaining pines in the mountain where the survey was done have developed tolerance to B. xylophilus.