The Invasive Polyphagous Shothole Borer Beetle, Its Fungal Symbionts and the Fastest, Most Intense Summer Experiment Ever…

One thing I’ve learned while in a teaching focused position: is that you have to collaborate with others, piecemeal together workspaces, and work fast and hard if you want to do research during the academic holidays. The fast part means designing experiments that will start and end within a reasonable time frame.. say- how about less than 2 weeks?

So I tried this approach.. and just wrapped up an intense 11 day experiment (12 days if you include clean-up time) that included three car trips back and forth between USC and UCSB, countless hours spent in a laminar flow hood, and the trips speckled with adventures and good times w/ friends and my collaborators.

In a nutshell, I collaborated with the UCSB Rivr Lab group to investigate the growth of the pathogenic invasive fungus, Fusarium euwallaceae, on different host plants- including two native willow species, two native cottonwood species and three avocado cultivars (Zutano, Bacon and Hass). (See funding and acknowledgements below this post!)

This invasive fungus causes Fusarium dieback disease on a lot of native plants in California, and is known to infect avocados (luckily there are management practices available to control the spread and pathogenicity in avocado orchards).

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This fungus is spread by the invasive polyphagous shothole borer beetle which was first discovered in Southern California in 2003, and is thought to have been accidentally introduced via products and/or shipping materials from SouthEast Asia. By 2010, this beetle was recognized as being the main cause of death of several street trees in Long Beach and by 2012 it was found in a residential backyard avocado tree. If you like your guacamole.. you can understand why the alarm bells started to ring!! This beetle has now been found in almost all Southern California counties and has been found as far north as Santa Cruz county. Furthermore, this beetle has invaded many other countries all around the world.

The spread of this beetle is problematic because it carries several mutualistic fungi (including Fusarium euwallaceae) that help it digest the tree tissues as it bores into its host trees. As the beetle bores into the tree and chews away- the spores of these fungi get released into the tree (beyond the protective bark) and can cause pathological symptoms in the tree, including ultimate death. Meanwhile the little beetles keep munching away and reproducing as much as they can.. and their offspring continue the cycle along with their mutualistic fungi …

Hence for management purposes- if we want to understand what tree species will be most impacted by this invasive beetle and its fungal symbionts- we need to know: 1) what species of trees are most likely to attract and be attacked by the beetle- and of these, which ones result in the highest survival, growth and reproduction of the beetle (and why)? and 2) what species of trees promote or prevent the growth of the different fungal symbionts (and why?), with a focus on Fusarium euwallaceae, which is particularly pathogenic. These questions are intertwined because the fungus needs the beetle to drill the holes for it, and to introduce the spores into the tree tissues; and the beetle needs the fungus to be able to digest the tree tissue to obtain nutrients and to keep growing, reproducing, etc.

There are many different research groups in California (including Akif Eskalen’s group at UC Davis, Shannon Lynch at UC Santa Cruz and Richard Stouthamer’s group at UC Riverside) and all around the world working on answering these questions. Our work this summer was focused on a small part of the larger picture, specifically investigating the growth of Fusarium euwallaceae on native California trees that grow in riparian habitats, as well as on several avocado cultivars. In regards to the native riparian plants, a graduate student at UCSB -Shelley Bennett, sampled from different parts of the Santa Clara River so that we could examine how proximity to the river impact the moisture, density and nutrients in the tree tissues and whether this impacts the fungal growth rate on these different host tree species.

Even though this was an 11 day experiment- it actually took a lot of prep work, a lot of troubleshooting and a lot of reconfiguring and pilot studies prior to execution of the actual experiment (what experiment doesn’t?!). Also complications due to Covid19 definitely put a huge slowdown on the research with massive supply and shipping delays and errors in June through July- which then meant that we no longer had time to cultivate the fungus from the beetles themselves (which requires cultivating, isolating and then propagating .. and ensuring that we had the right Fusarium isolates)- so we were very fortunate enough to receive fungal isolates from Richard Stouthamer’s lab at UCR just in time to start and finish our experiment before I started teaching classes (next Monday- eek!).

In the end we settled on two experimental methods:

1) using a saw to create saw dust (very inefficient I know) from over 140 branches of different host plants in order to produce unique host-plant autoclaved agar-based solutions that we could pour into petri dishes (along w/ just PDA agar replicates as a positive control); and 2) sawing cross-sections of the different branches, and bleaching, and rinsing these thin cross section pieces before putting into petri dishes (not-autoclaved). Then we put a 6 mm agar plug of Fusarium euwallaceae on the center of each of these samples (or a 6 mm agar plug without the fungus as a negative control).

The idea was to test the impacts of nutrient differences among these different host tree species (present in the agar-based solutions and in the cross-section pieces), and chemical defenses (which would ideally still be present in the cross-section pieces) in respect to the growth of the invasive fungus, Fusarium euwallaceae. Originally we did not want to use cross-sections, we wanted to create longitudinal slices.. but turns out that it is very difficult to do that safely and to get it to fit into petri dishes. So hence we had to change our experimental design on the spot. During the set up of this experiment, I also found out that I get very excited about the idea of consolidating nature from large sizes to small consolidated items that fit into a petridish.. is that weird?

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Anyhow- we just took this experiment down this past weekend- and now we have probably about 1000 images to process with imageJ (measuring the area of fungal growth from days 0-11) and over 140 spore suspensions to count with a hemocytometer (they are safe and sound in the freezer at this point .. I hope they are safe anyhow…). Wish us luck!

Since everything is now in a ‘do it later’ state- this experiment model gives me time now to finish prepping for my two classes (Env. Studies and Ecology) that start on Monday (eek!).. all online, with guided outdoor-distanced projects for my Ecology lab section- some as distanced as Taiwan!. I had to make an insect collection care package for that student.. Anyhow- stay tuned for my ideas on fun, distanced/remote hands-on learning for Ecology and Env Studies!

Funding and Acknowledgements

This work was funded by the Thelma Hansen Fund of UC ANR and additionally supported by Annemiek Schilder, the director of the Hansen Agric Research & Education Center in Santa Paula, CA. We want thank Dr. Richard Stouthamer (UC Riverside), his postdoc: Valeh Ebrahimi and lab technician Taha Farooqi for providing beetles and fungus during the initial phase of this research, as well as USC for use of their unused teaching lab space to conduct pilot studies, Ryoko Oono (UCSB) for her advice and use of her lab’s laminar flow hood, Carla D’Antonio for use of her lab space, and Dr. Akif Eskalen (UC Davis) and Annemiek Schilder for their advice. Huge shout out to Dr. Tom Dudley and Dr. Adam Lambert for this research opportunity and to Zoe Wood and Shelley Bennett for being awesome rockstar teammates on all of the field and lab work (Adam helped in the field too!)! I think their thumbs are still hurting from all of the sawing… lol (funny but maybe not funny).