Aerial spore sampling to assess infection risk and inform proactive management for root disease in ponderosa pine forests

Photo Credit

USDA photo by Patrick Bennett

An array of petri dishes containing freshly cut ponderosa pine wood disks. These disks were exposed to the air to trap Heterobasidion spores and measure deposition rates in ponderosa pine stands.

Heterobasidion irregulare is a fungus that causes root rot and mortality in ponderosa pine forests in the western United States and poses a serious risk to ponderosa pine silviculture. Heterobasidion root disease (HRD) has been noted as damaging in low-elevation, dry sites where ponderosa pine is often the only commercial species present. H. irregulare spores are produced from fruiting bodies that form on decaying stumps and infected trees. Freshly cut stump surfaces are the primary infection courts. Once spores infect a stump, the fungus can colonize the root system and infect healthy ponderosa pine trees at root contacts, thus increasing disease incidence in a stand and disrupting long-term management objectives. The fungus can remain viable for decades in underground woody biomass.

Spore infection can be prevented by treating stump surfaces with a borate compound post-felling. These treatments are highly effective, but application introduces extra cost and logistical complexity to timber harvest. This project will deliver tools and protocols for quantifying aerial spore loads and spore deposition rates for detection and monitoring of HRD to assess disease risk in ponderosa pine stands. Molecular techniques will be applied to detect H. irregulare from aerial spore samples and existing infections in stumps and live or dead standing trees.

Photo Credit

USDA photo by Patrick Bennett

Rotating spore impaction device for collecting airborne spores of Heterobasidion. The plastic case contains a battery and controls for powering a spinning motor contained within the PVC pipe assembly. The horizontal sampling arm holds two stainless steel rods that are coated with silicone grease to trap spores from the air as they spin.

Aerial spore density directly influences the spore deposition rate, and the spore deposition rate is strongly correlated with infection frequency. It would be very useful to have a monitoring protocol to assess infection risk. Where aerial spore densities are very high, it would be assumed that spore deposition rates and thus infection risk would also be very high. Trapping spores on wood disks is commonly used to detect and quantify the spore deposition rate of Heterobasidion, but it has seldom been used in ponderosa pine stands. Recent advances in molecular detection and quantification techniques, such as real-time PCR (qPCR) offer a highly sensitive, precise, and efficient alternative and complementary approach to quantifying aerial spore concentrations.

These detection and monitoring techniques will enable the development of site-specific risk assessments for HRD based on H. irregulare inoculum availability as well as existing disease incidence. This information will be used to guide disease prevention strategies such as post-harvest stump treatments and appropriate timing of harvest based on variable spore loads at different times of the year or under different environmental conditions. This work will also enable future monitoring studies that seek to answer questions about the fundamental biology of H. irregulare and its behavior in ponderosa pine stands in the Pacific and Inland Northwest (PINW). Future impacts of climate change including warmer and drier conditions may increase the susceptibility of ponderosa pine to infection, reduce its capacity for defense, and exacerbate the symptoms of HRD.

Approach

Researchers will use and compare two complementary spore trapping approaches to assess their relative strengths and weaknesses for use by land managers for monitoring H. irregulare spore loads and deposition rates:

Photo Credit

USDA photo by Patrick Bennett

Fruiting bodies of H. irregulare removed from a decaying ponderosa pine stump at one of the study sites in south-central Washington.

1. Rotating arm impaction spore sampler (aka rotorod): These traps consist of a small motor that drives a rotating arm with sampling rods that trap spores from the air. The rods are coated with silicone grease so that the spores stick to the rod surface. The spores can then be harvested by dissolving the silicone grease into a liquid solution. This liquid spore suspension can then be subjected to DNA extraction and qPCR with H. irregulare specific primers to quantify spore loads.
2. Wood disk exposure method: This method involves exposing fresh ponderosa pine disks to the air, incubating them in the lab, and quantifying spore deposition by counting colonies from Heterobasidion spores that have germinated on the disk surface. This method has the added benefit of assessing spore viability, as only spores that germinate would be counted.

The methods described above can be used to estimate the aerial spore density (spores/m3/hour) and spore deposition rate (spores/m2/hour). These spore trapping methods are being deployed simultaneously in ponderosa pine stands.

Molecular techniques will also be applied to detect H. irregulare in stumps, standing dead trees, and live trees. Increment borers and/or drills will be used to extract tissue at four quadrants around the bases of trees and/or stumps. Wood cores or drill shavings will be subjected to DNA extraction, and PCR will be performed with species-specific primers to detect H. irregulare in the samples with a high level of sensitivity and accuracy.

These complementary methods could be used for routine monitoring and detection of H. irregulare in ponderosa pine stands and provide a variety of tools that can be used to address the various management needs and goals of the users. For example, these techniques will be useful for establishing an action threshold (e.g., minimum aerial spore concentration) to help land managers decide when preventative management actions should be taken.

Deliverables

The tools will be shared via virtual and/or in-person workshops and field demonstrations. Toolkits will be developed and delivered to land managers. These kits will include sample collection materials, aerial spore trapping devices, and other equipment that can be loaned out to users. A printed guide will also be produced for land managers that details the protocols for monitoring and detection of HRD.