Many Forest Invasive Alien Species (FIAS) are threatening Canada’s Forest. In the bioSAFE Project, we will be researching two pests and two pathogens.

The bioSAFE project is taking a two-fold approach in the development of its biosurveillance tools. The approaches being used are: (1) determine the global distribution of the organism in order to map out the likely source of the pest or pathogen, and (2) analyze the functional characteristics that allow the pest or pathogen to spread and survive in their non-native locations.

The key in the second approach is to identify markers of Fitness and Outbreak-Related Epidemiological (FORE) traits in forest insect or pathogens outbreaks which represent a highly valuable goal for current and future risk mitigation efforts and monitoring. There are some traits that can be clearly associated with invasiveness and could be useful in predicting some aspects of outbreaks. Virulence and host range in pathogens, and flight capacity and overwintering capacity in insects have been related to global outbreaks or risk of epidemics and are likely to be heritable and, therefore, amenable to genetic and genomic analyses. These traits are likely to be genetically complex and will require multiple approaches to find associated markers with sufficient predictive power.

Here is a basic introduction of the four organisms the bioSAFE project is tackling and what our scientist will be studying.

 

Asian Longhorned Beetle (ALB)

Asian Longhorned Beetle (ALB)
Asian Longhorned Beetle (ALB)

Asian longhorned beetle (ALB) is a wood-boring insect native to China and Korea. ALB attacks nearly all broadleaf trees in North America, but prefers native maples. Females lay eggs through the bark and larvae tunnel through the living tissue interrupting water and nutrient transport, killing the tree. ALB is easily transported in firewood, live trees or untreated lumber wood such as packing material used in shipping. 

ALB was first introduced to North America in the 1990s, probably in solid wood packaging materials, such as crates or pallets. In 2003, ALB was discovered for the first time in Canada in an industrial park in the Toronto area. The infestation was controlled by removing host trees within a quarantine zone around the infestation to prevent further spread, at a cost of tens of millions of dollars over four years. Another ALB infestation was detected in Toronto in 2013. Eradication and detection efforts were initiated for a second time, and the area is being actively monitored to determine their success. Overall, more than US$500M has been spent on ALB eradication and associated monitoring in North America since the 1990s.

Global distribution

ALB have a broad native range in China, and we expect that some of the populations will be more likely to be able to overwinter in harsh Canadian winters. If we can identify those populations, it will allow resources to be prioritised towards the riskiest introduction events. One part of the BioSAFE team (led by Dr. Ilga Porth, Université Laval and Dr. Amanda Roe, Natural Resources Canada) is identifying genomic markers called single nucleotide polymorphisms (SNPs) to allow us to effectively identify the geographic source of a new infestation.  

FORE traits

A parallel, related, approach is led by Dr. Brent Sinclair (Western University), Dr. Roe and Dr. Daniel Doucet (Natural Resources Canada), who aim to find genomic markers (Figures 1 and 2) that indicate the ability of a given ALB to survive winter. To do this, they are first determining the physiology responsible for overwintering success, and then using transcriptomics and metabolomics to identify the pathways and molecules that underlie those physiological mechanisms. They will then use the SNP database to identify SNPs that are likely to be speciically associated with enhanced overwintering capacity. This work is being performed at the natural resources Canada Insect Production and Quarantine Laboratory (IPQL) - the only Canadian facility certified to house ALB colonies.

Figure 1.

 

Figure 2

 

Flow charts by Dr. Alex Torson

Dutch Elm Disease (DED)

Dutch Elm Disease (DED)
Dutch Elm Disease (DED)

Since its introduction from Europe, Dutch elm disease (DED) has destroyed millions of elm trees across North America. The pathogen is spread by native and introduced bark beetles whose larvae tunnel under the outer bark of elm trees and create distinctive feeding galleries. The adult beetles spread the fungus into live trees, resulting in fungal proliferation in the trees’ tissues. The first introduction of DED was reported in 1930 and the disease reached Eastern Canada in the 1940's and Manitoba in 1975. The advancing front is now in Saskatchewan but the disease does not occur in Alberta or British Columbia. 

At least two pathogens, Ophiostoma novo-ulmi and O. ulmi cause the disease and have different virulence. These pathogens belong to the ophiostomatoid fungi, a group that includes other dangerous and quarantined pathogens such as oak wilt (Ceratocystis fagacearum), Japanese oak wilt (Raffaelea quercivora) and the black stain root disease of conifers (Leptographium wageneri). These fungi are vectored by beetles and can be easily transported in firewood and lumber. These fungi are of phytosanitary concern, and are monitored and managed by the CFIA. Intensive surveys are conducted annually in provinces on the advancing front, as well as in provinces where the pathogen is established.

Global distribution

To reconstruct the global outbreak of DED we are going to analyse the genomic data of historical DED samples collected over many years. We will put together a historical evolutionary map based on the chronology of this global outbreak which will give us a unique look in the evolution and adaptation of DED and possibly other pathogens that have global outbreaks.

FORE traits

Understanding what characterizes tree pathogens that cause large-scale outbreaks can provide predictive analytical and detection tools. The Dutch elm disease pathogens vary from highly virulent (O. novo-ulmi) to moderately virulent (O. ulmi) or mildly virulent (O. himal-ulmi depending on the host). We will search for virulence determinants in this disease using a genomics approach and develop markers predictive of virulence. Different methods to identify genomic regions associated with virulence in non-model plant pathogens will be applied to DED. We will do this by identifying candidate genes associated with virulence, temperature adaptation and substrate utilization. We will match the phenotypes against the variants seen in the genome sequences of the collection of DED samples.

Sudden Oak Death (SOD)

Sudden Oak Death (SOD)
Sudden Oak Death (SOD)

Phytophthora ramorumis an oomycete (a group of fungus-like microorganisms) that can attack over 100 host species, including several of our most valued tree species in Canada such as Douglas fir, larch and oaks. The most damaging diseases caused by this pathogen are sudden oak death (SOD) in California and Oregon and sudden larch death in England. This pathogen has killed hundreds of thousands of trees, affected landscape processes, and caused economic hardship in North America and Europe. The disease was discovered in nurseries in British Columbia in 2003, placing Canada’s forests at risk. Since this discovery, aggressive eradication and phytosanitary certification programs have helped contain the pathogen to a few nurseries in BC. The CFIA conducts inspections and annual surveys to identify the species and lineages using a combination of culturing of the pathogen and DNA assays developed through foundational research by this team.

Current biosurveillance techniques cannot differentiate between novel introductions and existing infections that escaped eradication efforts in nurseries experiencing repeat occurrences. Differentiating between these scenarios will guide CFIA control measures. Available survey methods are also unable to reliably assign a pathogen sample to a source population. Biosurveillance of SOD is further complicated by their ability to hybridize, rapidly evolve, and in some cases jump hosts. None of these pathogenic innovations can be accurately monitored using existing biosurveillance and detection methods, so a comprehensive genomics approach would help address these challenges.

Global distribution

We have conducted a retrospective genomic analysis of more than 500 samples of P. ramorum from Europe, the USA and Canada over multiples years to build an exhaustive database of genomic profiles of P. ramorum. This will serve to conduct a genomic epidemiology analysis to track the Canadian SOD outbreak and identify sources and pathways and to develop the Phytoseq target enrichment tool. We will assess mitigation efficacy by comparing the genomic profiles of SOD isolated before and after eradication. The Phytoseq tool allows the accurate and rapid identification of single nucleotide polymorphisms (SNPs) that discriminate the different lineages of P. ramorum and also identifies intralineage polymorphisms that will be informative in epidemiological applications. The Physeq tool can be used directly from infected tissue, making it a useful tool for large scale genomic biosurveillance.

 

Figure 1. Amplification and sequencing of targeted genome regions directly from infected plant tissues and accurate assignment to lineages of Phytophthora ramorum.

FORE traits

Phytophthora ramorum possesses two traits that make it a remarkably dangerous pathogen for Canada’s forests and trees: the ability to attack hundreds of plant species and the ability to attack woody tissues. These characteristics are of particular concern to the international phytosanitary community since Phytophthoras possessing similar traits could be disseminated on wood products. We are comparing phylogenetically related Phytopthora species with contrasting traits: multi host vs single host, weak vs aggressive attacks, woody tissues vs foliar. This allows us to analyse their biological and genomic profiles to discover gene families that increase the pathogenicity of the species. We are currently analysing gene expression in the different P. ramorum lineages and finding distinct gene expression profiles (Figure 2).

 

Figure 2. Phytophthora ramorum lineages exhibit unique gene upregulation in Rohododendron leaves

Asian Gypsy Moth (AGM)

Asian Gypsy Moth (AGM)
Asian Gypsy Moth (AGM)

Asian gypsy moth (AGM) is a lepidopteran regulated in North America and Europe, and is one of the most threatening FIAS in Canada and the USA. It has a broad host range, attacking most species of deciduous and coniferous trees in Canada. Female AGM have a strong flight capacity and can lay eggs on any surface; egg masses are often found on container ships. An incursion of AGM in Canadian forests could affect not only the forestry and nursery sectors, but also other economic activities. For example, the shipping industry must ensure that vessels entering Canada do not carry AGM egg masses. When ships carrying goods are found non-compliant, they are turned away from Canadian waters, interrupting the flow of goods at the cost of the shipping company, affecting downstream industries that depend on product delivery, leading to further economic losses. 

Keeping AGM out of Canada requires inspection of vessels entering the country to ensure that they are free of egg masses and requires biosurveillance surveys around ports of entry to detect adults. When egg masses are discovered on non-compliant vessels it is important to identify them to determine which species they belong to and to ascertain their potential sources. The CFIA requires information about the origin of intercepted insects to improve phytosanitary procedures, regulations and compliance of shipping companies, and identify countries that represent higher risks. This information guides technical negotiations with our trading partners. Biosurveillance requires accurate identification of AGM at all life stages of the insect. Flight capabilities also differ between the established gypsy moth and the AGM but are hard to discern between hybrids. A molecular method to rapidly detect hybridization and predict flight capability in gypsy moth would guide CFIA management decisions.

Global distribution

To develop a reference for tracking sources and assessing the level of hybridization in natural populations, we conducted, in 2017 and 2018, a massive field collection campaign targeting the gypsy moth over its entire geographic range (85 collection sites; 26 countries), for the purpose of generating genomic profiles. We are now processing these data to (i) characterize gypsy moth global population genetic structure, (ii) identify hybrid zones, and (iii) select genetic markers that can be used for source identification of intercepted specimens.

FORE traits

Flight capacity is an important trait for invasive insects. In contrast to the European gypsy moth (EGM), AGM females are strong flyers, thereby facilitating spread, and the species mate to produce hybrids with varying degrees of flight capability. Introgression could generate individuals with increased flight capacity, in a largely European genetic background. Our experiments will search for candidate genes associated with flight capacity by looking at populations of parents and their progenies that will be functionally validated.

Host utilization in European and Asian gypsy moth is another important FORE trait. We have tested Asian gypsy moth (AGM) feeding on 10 most important Canadian conifers, i.e. Pinus strobus, P. contorta, Picea glauca, P. mariana, P. abies, Pseudotsuga menziesii, Tsuga canadensis, Thuja plicata, Abies balsamea, and Larix laricina, respectively, at the containment facility in Connecticut (USDA) (our collaborator: Dr Melody Keena). We obtained information on conifer host preference for the AGM strain based on: (a) most larval weight gain on a host, (b) highest survival rate on a host, and (c) fastest development to the 4th instar within 10 days. We performed RNAseq on larval midgut tissue and a first analysis suggested several important detoxification genes in AGM are upregulated. In order to obtain an inventory of pre-existing biochemical host defenses (constitutive), we sampled prior to feeding host tree foliage and ran the analytics for phenolics and condensed tannins content (Figure 1). Our results suggest important variation in host biochemistry among conifer species. We are currently in the process of analyzing foliage terpenes as well. Finally, we will be able to interpret our findings on gypsy moth detoxification potential and provide directives which conifer species are indeed most susceptible in Canada. This work is co-directed by Dr Ilga Porth (Université Laval) and Dr Christopher Keeling (Canadian Forest Service).

This work is also featured on the website of the Quebec-based 2RLQ research partnership (Réseau Reboisement et Ligniculture Québec, under research activities, to promote research and development, knowledge transfer, and networking in the area of plantation forestry.

Figure 1 & 2 

  

Figure 1, work and photo credit: Mr Loic Soumila (PhD student), collaborators: Dr Almuth Hammerbacher (MPI Jena), Dr Peter Constabel (University of Victoria)

 

What's new
in the bioSAFE project