Host association and evidence of geographic isolation of the genus Onnia in North America and the development of a molecular tool for field detection

Abstract

Tomentosus root rot, a disease found in coniferous forests throughout the northern hemisphere, is caused by Onnia spp. This disease causes growth reduction, root decay, and weakens trees, which increases the likelihood of mechanical failure. The genus has a wide geographic distribution, and this distribution, alongside a substantial reduction in growth of infected trees, makes Tomentosus root rot a highly impactful disease for timber operations. However, management of this disease is difficult because these saprophytic fungal pathogens can persist in stumps and other decaying organic matter making it impossible to eradicate. The aboveground symptoms caused by Onnia spp. are common for most root diseases, which makes diagnosis challenging. This difficulty in identification and persistence in a stand brings challenges to managing this disease for both large-scale operations and recreational sites such as campgrounds. Due to recent taxonomic changes, the geographic distributions and host ranges of several North American Onnia spp. is not well understood. A clear understanding of host association and species distribution is important for developing management strategies. Quick and accurate identification of these pathogens would enable early detection and a proactive response for management. Therefore, this research had three objectives to better characterize these pathogens and develop tools for the detection and identification of Onnia spp. in North America: 1) provide an overview of the existing literature regarding the host associations, geographic distributions, and molecular tools available, 2) conduct surveys and use DNA-based identification techniques to describe the host associations and geographic distributions of North American Onnia spp., and 3) design and validate a rapid field-based molecular detection assay for North American Onnia spp. Chapter one is a literature review that consolidates and summarizes previous research and knowledge on the genus. Historical accounts of Tomentosus root rot are obfuscated by numerous changes in genus and species classifications and the debates that followed. As such, making confident inferences on the distribution, host association, and management strategies are difficult. However, what is clear is that historically, two closely related species that both produce a white pocket rot and similar basidiocarps were recognized in North America. These were referred to as O. tomentosa (= Inonotus tomentosus) and O. circinata (= I. circinatus). A defining feature that was used to distinguish these species was the hymenial setae, which were either hooked (O. circinata) or straight (O. tomentosa). However, investigations using molecular tools showed that the species that exhibited hooked hymenial setae comprised two distinct species, O. subtriquetra and O. leporina. The fact that O. subtriquetra and O. leporina were considered the same species has resulted in a misunderstanding of their actual geographic distributions. There has also been some doubt cast on the distribution and host association of O. tomentosa. This is because the main distinguishing feature between O. tomentosa and the other two species is that it has straight hymenial setae. This feature requires the use of a microscope for identification and there is some concern that previous reports of O. tomentosa were based solely on the existence of basidiocarps without further investigation of the morphology of the hymenial setae. In Chapter two, 58 isolates of Onnia spp. collected during surveys, along with those borrowed from herbaria, were identified using DNA sequencing. We found evidence of three species of Onnia spp. in North America: O. tomentosa, O. subtriquetra, and O. leporina. O. subtriquetra appears to be primarily a pine specialist found from as far west as California to the Carolinas. We confirmed O. tomentosa as a widespread generalist across North America and discovered evidence that the species can be separated into three lineages, there is no evidence of recombination across the lineages. However, a neighbor-net phylogenetic network showed some evidence of reticulation among O. tomentosa lineages, suggesting that there is a soft barrier that prevents random recombination among lineages, but some gene flow may be present. Unfortunately, few inferences could be made regarding O. leporina, as only a single specimen, from Quebec, Canada was available. Chapter two also describes the development and validation of a set of primers for a loop mediated isothermal amplification (LAMP) assay. This molecular assay requires little training, equipment, and reaction time, making it a valuable tool for land managers or forest pathologists who need an inexpensive and rapid technique for diagnosing infected trees. The efficacy of the LAMP was determined by testing it against 30 North American Onnia isolates as well as other co-occurring fungi (e.g., Armillaria spp., Coltricia spp., Porodaedalea spp., Phellinus spp., Fomitopsis spp., Heterobasidion spp., and Coniophora puteana). The limit of detection for this molecular test was 170 pg of DNA for O. tomentosa isolates, 83 pg for O. subtriquetra, and 121 pg for O. leporina. We demonstrated the field applicability of this assay and found that temperature fluctuations of premixed reagents increased the likelihood of false positives. This molecular tool will allow for reliable detection and identification of Onnia spp. in North America even when white pocket rot is the only indication of infection, which will aid in the diagnosis and management of Tomentosus root rot.