Higher foliar defensive compounds (terpene) concentration and significant variations of species abundance found in fungal endophytes across Canada: implications for a biological control agent of spruce budworm

Haolin Wei

Abstract

Fungal endophytes can be a possible biological control agent for spruce budworm as some species indirectly promote the biosynthesis of plant defensive compounds (terpenes). And those defensive compounds can reduce the damage of spruce budworm. White spruce (Picea glauca) is a national coniferous tree species distributed across Canada (Rweyongeza et al., 2007). It contributes to millions of dollars in forest sector income and takes up 45.9% of volumes of coniferous tree species in Alberta (Rweyongeza et al., 2014). Spruce budworm (Choristoneura fumiferana (Clemens)), which is outbreak every 30 years, is one of the most severe forest pests in Canada and causes millions of cubic meters of loss in white spruce (Chang et al., 2012). Fungal endophytes are harmless symbiotes to tree species that induce the release of some secondary metabolites (Stefani & Bérubé, 2006). Terpenes are secondary metabolites that can reduce the attack of spruce budworms (Lirette & Despland, 2021).

Moreover, the knowledge of the abundance of fungal endophytes in white spruce (Picea glauca) across Canada is not well-known. So, it is essential to know the abundance of white spruce (Picea glauca) fungal endophytes across Canada and the relationship between terpenes and fungal endophytes, as these can provide the baseline of future forest management practices and the potential of a biological control agent for spruce budworm (Stefani & Bérubé, 2006). The fungal endophytes can be applied by spraying, stem injecting, root dipping and seed soaking into white spruce trees as a biological control agent (Mantzoukas & Eliopoulos, 2020).

This study investigated the variation of foliar terpenes and fungal endophytes abundance and the relationship between fungal endophytes and terpenes. Thirty 20-30 years old white spruce trees from six locations among five provinces are selected as the study sample. Terpenes concentration is measured via Gas Chromatograph/Mass Spectrometer (GC/MS) using fresh phloem in each tree. The abundance of fungal endophytes is measured via DNA from foliar tissue collected from the tree stem's tip. DNA is extracted using the EZNA ® Fungal DNA Kit. Foliar DNA is concentrated by a two-step polymerase chain reaction (PCR). The final product is analyzed via the Illumina MiSeq platform in the Molecular Biology Facility at the University of Alberta. The abundance of fungal endophyte genus is measured by the number of amplicon sequence variants (ASVs) reads. The amplicon sequence variants (ASVs) are single fungal DNA sequences that can be used to classify fungal taxa and measure relative abundance (Beule et al., 2021).

Saskatchewan has the highest abundance of total fungal endophytes, followed by Alberta. In comparison, New Brunswick has the lowest abundance of total fungal endophytes. Fungal endophyte genera Cladosporium is abundant in every province but more abundant in Alberta, with over 3700 ASVs. Genera Lirula is plentiful in Saskatchewan, with over 4000 ASVs. No variation in terpene is found, but Saskaetchawen and Alberta's sites have higher total concentrations of it. In addition, six fungal endophyte genera: Lirula, Chalara, Pleurotus, Periconia, Phialocephala, and Gnomoniaceae, are found to positively correlate with total terpene (monoterpene/sesquiterpene) concentration. No correlation is discovered between total fungal endophyte abundance and concentration of any detected terpene.

Figure-1 White spruce distribution across Canada (Figure comes from USDA,1971)

Photo-1 Fungal endophyte Rhizosphaera kalkhoffii grows on white spruce needles (Photo taken by Bruce Watt, 2010).

Photo-2 Fungal endophyte genera Cladosporium grow in Petri dishes (Photo taken by Koul et al., 2017).