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Forest Health and Biotechnology: Possibilities and Considerations (2019)

Chapter: 3 Mitigating Threats to Forest Health

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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 57
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 59
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 60
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 62
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 63
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 64
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 65
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 66
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 67
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 68
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 69
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 70
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 71
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 78
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 79
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 80
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
×
Page 81
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 82
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 83
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 84
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 85
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
×
Page 86
Suggested Citation:"3 Mitigating Threats to Forest Health." National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. Washington, DC: The National Academies Press. doi: 10.17226/25221.
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Page 87

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3 Mitigating Threats to Forest Health There are multiple options for dealing with forest insect pests and pathogens, but feasibility and suc- cess vary widely. To assess the unique challenges and opportunities that a biotech tree may present as a tool for addressing forest health, it is important to understand the other options available. Given the spatial and temporal nature of forest health threats from insect pests and pathogens, it is also likely that a combi- nation of approaches might be needed to ensure proper management of an infestation. The most cost-effec- tive approach for protecting forest health from nonnative insect pests and pathogens (Finnoff et al., 2007) is to prevent introduction, followed by early eradication after arrival (Liebhold et al., 2016). Once estab- lished, the impact and cost of dealing with the infestation rapidly increase (Roy et al., 2014). Given that human mobility and trade volumes—major drivers of pest introductions—are likely to continue to rise, the enforcement and enhancement of preventive measures will become even more critical (Lovett et al., 2016). Even where prevention or eradication has been successful, forests will remain vulnerable to repeated intro- ductions of the same nonnative insect pests and pathogens over time. Once established and spreading in forests, whether pests are native or nonnative, multiple manage- ment options may exist. Management can focus on trying to minimize the damage and mortality to the forests (including the large, old-growth trees), on actively preparing to regenerate or restore a species, or on both strategies. If the impacts are not severe enough to alter the species’ ecological footprint or manage- ment actions appear unrealistic or undesirable, managers may decide that taking no action is the best alter- native. However, if the decision is to take action, the focus turns to early detection and response, contain- ment, and long-term management to restrict further expansion and impact (Liebhold et al., 2017). Management options include biological control and integrated pest management, and various forms of site management (e.g., pesticide use, containment, fire, thinning) (Liebhold et al., 2017). When outbreaks of insect pests and diseases affect only one or a few tree species, the larger impact of such pests is directly related to the dominance of the host species. Thus, maintaining high levels of diversity may be an effective management approach to minimize impact. In low-diversity forests, other approaches may be more im- portant. In many of the most extreme cases, because of the high susceptibility of native tree species to some nonnative insects and pathogens with substantial dispersal potential, it will not be possible to prevent ex- tremely high mortality in the affected tree species. Once an insect or pathogen is established, there are a number of key management tools that might be considered to retain the presence of the tree species in North American forests into the future. These include (1) the enhancement of genetic resistance, (2) the develop- ment and use of biocontrol agents, (3) the development and use of chemical control methods, and (4) man- agement practices to prevent or decrease the infestation. The enhancement and use of genetic resistance can proceed through the development and deployment of selective-resistance breeding within either the native species or from closely related nonnative species or the development and deployment of resistance using biotechnology. The effectiveness of these varied approaches to prevent and manage insect pests and pathogens varies across systems and infestations (Lovett et al., 2016). The time line for use of these tools in management activities for forest trees and forest health will depend on a number of factors, but the biology of the species involved (both tree and insect or pathogen) and the environments in which the tree species exist will have a major influence. Insecticides and fungicides are often used in attempts to preserve existing forest stands Prepublication Copy 51

Forest Health and Biotechnology: Possibilities and Considerations or to protect large individual trees in urban settings. They are usually expensive, may have negative envi- ronmental impacts, and in some cases provide only a stopgap measure to give time to consider or develop other alternatives or the hope that future environmental conditions become less conducive for the damaging insect or pathogen. The same can often be said of many biological control agents. This chapter provides an overview of the different approaches and the approximate time required for implementation. The case study species are featured to illustrate the differences between species and considerations of the merits of different approaches. PREVENTING INTRODUCTIONS Preventing the introduction of insect pests and pathogens yields the largest ecological and economic benefits (see Figure 3-1; e.g., Mack et al., 2000; GAO, 2015). International trade agreements include clauses aimed at reducing these introductions (Burgiel et al., 2006). These are being implemented by the Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA) and include quarantine, interception (e.g., inspection, decontamination), and pre-border treatments (e.g., fumigation, immersion, spraying, irradiation, extreme temperatures) at the point of origin and during shipment (Haack et al., 2014; Leung et al., 2014). The International Standards for Phytosanitary Measures Protocol 15 (ISPM-15) were developed under the International Plant Protection Convention to reduce the movement of wood-boring insects in pallets and other wooden shipping materials (Haack et al., 2014). Wood borers are among the most serious of insect pest invaders: 58 species of wood borers became established in the United States between 1909 and 2008 (Leung et al., 2014). The approved methods for wood treatment in the United States include heat treatment (conventional and dielectric) and fumigation with methyl bromide. An economic analysis by Leung et al. (2014) concluded that implementation of ISPM-15, though expensive and not fully effective, would save the United States more than $11 billion by 2050 in avoided impacts. These pre-border efforts are often coupled with the post-border protection efforts of inspection, quarantine, and treatment of imported mate- rials that facilitate interception of insect pests and pathogens prior to their potential escape. FIGURE 3-1 Stages through time of the typical process, extent of infestation, and control costs associated with the introduction of insect pests and pathogens. SOURCE: Adapted from GAO, 2015. 52 Prepublication Copy

Mitigating Threats to Forest Health EARLY DETECTION AND RAPID RESPONSE Early detection and response programs are essential to prevent the spread of introduced insect pests and pathogens, although these practices may not be effective against microscopic species (i.e., most patho- gens) (Liebhold et al., 2016). In addition, public awareness through educational programs may be instru- mental in minimizing the entry of harmful organisms and their early detection. Surveillance methods to facilitate early detection can include deployment of pheromone and other traps, monitoring of sentinel trees or vulnerable sites, and solicitation of reported sightings (Kalaris et al., 2014). Spatial modeling of locations of highest risk of invasion (Venette et al., 2010) can guide deployment of early detection efforts. Liebhold et al. (2016) reviewed both the uses of and methods for surveillance, ranging from baseline early detection to infestation delimitation, to verify the success of an eradication effort. Eradication Eradication (see Figure 3-1) is dependent on effective early detection efforts because eradication is more successful when introduced populations cover small areas (Liebhold et al., 2016). Success is also dependent on the detectability of the insect pest involved and whether species-specific control tools are available (Tobin et al., 2014). Chemical traps, mating disruption (e.g., releasing sterile insects), and insec- ticide fumigations can be used to eradicate small populations of insect pests. For example, pheromone traps have been deployed at the advancing front of the introduced gypsy moth (Lymantria dispar) for early de- tection of spreading populations that can then be treated (Sharov et al., 2002). For pathogens, mechanical removal of the infected host may be the only viable practice, given that detection of the pathogen may not be feasible before infestation. Overall, eradication can either remove or contain the threat or delay the spread of the insect pest or pathogen while more effective management methods are developed (Liebhold et al., 2017). Although the ability to eradicate pests has improved over time (Liebhold et al., 2016), many attempts have been unsuccessful. For example, eradication of white pine blister rust (Cronartium ribicola) to protect species of five-needle pine was a multimillion-dollar effort extending over more than 50 years in the 20th century. The principal approach was through removal of Ribes species (e.g., currants and gooseberries), the alternative host for the pathogen to complete its life cycle. However, this effort is regarded as a failure in the western United States (Maloy, 1997). As of 2018, it is generally acknowledged that white pine blister rust will have a permanent presence in North America. Emerald ash borer, first detected in Michigan in 2002, was found in Ohio and Maryland in 2005, indicating that eradication efforts were not effective. At- tempts at eradicating the emerald ash borer were unsuccessful in part because of unintentional long-distance dispersal in nursery stock, movement via infested firewood and vehicles, the long-distance dispersal ability of the insect, the difficulty in detecting the early stages of infestation, the absence of a long-range sex or aggregation pheromone, and the lack of a suitable attractant for mating disruption (Mercader et al., 2011, 2016; McCullough and Mercader, 2012). In other instances, eradication efforts have been constrained by negative public reactions to the methods used, such as removal of potential host trees, release of irradiated insects, or broad spraying of a pesticide (Liebhold et al., 2016). Further review of this literature can be found in Liebhold et al. (2016). CONTAINMENT AND LONG-TERM MANAGEMENT When eradication of a nonnative is not possible or the spread of a native or established nonnative pest is inevitable, a variety of management options may be pursued. One option is to take no action. Although the option to take no action often is the de facto outcome because the discovery of a new introduction or recognition of increasing impacts of a species already present lags the infestation of hosts (Liebhold et al., 2017), the committee defines “no action” as a deliberate management decision that is weighed against other options. Options to minimize the effects of insect pests and pathogens include (1) biological control, Prepublication Copy 53

Forest Health and Biotechnology: Possibilities and Considerations (2) site management practices (including applying the types of chemical, host removal, and sterile insect techniques also used for eradication), and (3) enhancement of genetic resistance through selective breeding, hybridization, or biotechnology. These options may be implemented independently or in combination. No Action The decision to take no action may result from a determination that the insect pest or pathogen is unlikely to have significant (further) impacts on individual species or forest health, a lack of resources, or an inability to identify an effective action to take. This last reason would likely inspire further research if significant impacts are anticipated. The decision to take no action recognizes that vulnerable tree species may decline or be lost entirely, with potential cascading impacts on other species and ecosystem services. The ecological effects will depend on the role of the tree species in the environment, whether replacement species fill similar niches, and whether replacement species are themselves later subject to pest outbreaks. The no-action decision may be made at the time that the host tree is threatened or when restoration (e.g., via breeding or breeding in combination with a biotechnology approach) is considered. Biological Control and Integrated Pest Management Biological control is the intentional introduction or application of populations of natural enemies or competitors to control insect pest species (Kenis et al., 2017). Two types of biological control have been effective against introduced insects. The insect pest itself can be manipulated to reduce population growth (e.g., release of sterile males to suppress population growth by competing with fertile males). Alternatively, if the lack of natural enemies (i.e., enemy release) in the new range is the major driver of the outbreak, specialist natural enemies can sometimes be identified in the indigenous range of the introduced insect pest species and released into the area of invasion (Liebhold et al., 2017). Biological control can be non-self-sustaining, such as when large numbers of sterile males of the insect pests are released, inundating the population sufficiently to dominate breeding, thus reducing the growth of a pest population. In this case, the released organisms are not self-sustaining in the environment, so this approach requires release whenever population control is necessary. Self-sustaining biological control methods include introduction or augmentation of natural enemies that reproduce and are maintained in that location without successive applications. For North American trees, these include control of species such as the winter moth (Operophtera brumata) and the larch case bearer (Coleophora laricella) with parasitoid insects introduced from overseas or from another region of the North American continent (Wainhouse, 2005; Kenis et al., 2017). Management practices that favor native predators of the insect pests (conservation biological control), such as providing shelter and alterna- tive food sources for those species, are also common strategies to minimize the likelihood of damage from insect pests (Tscharntke et al., 2007). Overall, a review of biological control in the United States concluded that establishment of biological control agents targeting insect pests of trees has been more successful than those targeting pests of herbaceous species (Kenis et al., 2017); still, the success of these programs can be quite variable (see Box 3-1). Both Liebhold et al. (2017) and Kenis et al. (2017) provide reviews of biolog- ical control efforts to reduce the impacts of insect pests on trees. Hypovirulence is a biological control strategy for mitigating or suppressing the effects of some fungal pathogens. Some viruses can infect pathogenic fungi, reducing their ability to infect, colonize, kill, and reproduce on susceptible hosts (Boland, 2004). If these viruses are spread in the area infested with a prob- lematic pathogen, in some instances they may reduce the virulence of the pathogen of interest. Success with this strategy has been demonstrated in Europe on strains of Dutch elm disease (Boland, 2004) and chestnut blight (see Box 3-1; Grente and Sauret, 1969; Grente and Berthelay-Sauret, 1978). 54 Prepublication Copy

Mitigating Threats to Forest Health BOX 3-1 Biological Control Efforts and Site Management Practices in Case Study Species American Chestnut As mentioned in Chapter 2, site management practices such as chemical treatments and clearing and burning were ineffective in controlling chestnut blight in the early 20th century (Stoddard and Moss, 1913). Biological control via hypovirulence, however, shows some potential because the European chestnut (Castanea sativa) also suffered severe damage from the chestnut blight fungus in the early 20th century but recovered substantially due to the emergence and deployment of hypovirulent strains of the fungus (Grente and Sauret, 1969; Grente and Berthelay-Sauret, 1978). Trees infected with Cryphonectria parasitica, which in turn are infected with the hypovirus, show restricted canker develop- ment and are able to continue growing and reach maturity (Jacobs et al., 2012). In contrast to the results in Europe, however, the hypovirulent fungal strains in North America have limited ability to spread from tree to tree and spread much more slowly than the uninfected fungus; therefore, American chestnut populations are not protected (Anagnostakis and Hillman, 1992; Milgroom and Cortesi, 2004). The spread of the hypovirus depends on fusion (anastomosis) of the hyphal filaments (mycelia) that constitute the vegetative growth of the fungus. The vegetative structure formed by the hyphae is known as the mycelium. The failure of the hypovirus has been attributed to multiple genetic variants of the “wild type” fungi that express different vegetative incompatibility (vic) genes. If the vic genes match, fusion of hyphae may occur. If the vic genes do not match, fusion of hyphae is blocked and the hypovirus is not transferred. Therefore, the spread of hypovirulence from tree to tree is blocked by mycelial incompatibility (Liu and Milgroom, 1996; Milgroom and Cortesi, 2004). It is assumed that there are a larger number of mycelial incompatibility groups in North American fungal populations than in Europe (Liu et al., 2002). Genetic analysis has identified six diallelic vic loci (loci with two alternative vic alleles) regulating vegetative incompatibility (Cortesi and Milgroom, 1998). These genes have been identified at the mo- lecular level (Choi et al., 2012) and have been disrupted using an adapted Cre-loxP recombination sys- tem resulting in the loss of the incompatibility barriers. The results demonstrate the feasibility of a “super” hypovirus that could overcome the genetic incompatibilities and transmit a virulence-attenuating hypovirus for biocontrol of the chestnut blight fungus (Zhang et al., 2014; Zhang and Nuss, 2016). Hypovirulence may yet become useful in combination with host resistance for biocontrol of blight (Griffin, 2000). Whitebark Pine A limited number of studies have used fire or fire-surrogate treatments (thinning, fuels enhancement, or small selective cuts to encourage nutcracker caching) to investigate efficacy at increasing regenera- tion of whitebark pine. High- and moderate-intensity prescribed fire treatments along with selective cut- ting combination treatments were successful in creating nutcracker caching habitat; however, few to no whitebark pine seedlings had established after 5 years (Keane and Parson, 2010). This lack of regener- ation, even when nutcracker caching was high, may be due to many factors, and the assessment time frame may have been too short to detect effects because whitebark pine may take decades to reestab- lish (Arno and Hoff, 1990; Tomback et al., 2001). It may also be possible that, in areas where high mortality of cone-bearing trees has occurred, nutcrackers recover most seed caches for food, leaving few to no seeds to germinate (McKinney and Tomback, 2007). Furthermore, studies have revealed that prescribed fire often kills many mature whitebark pines while the numbers of competing subalpine fir targeted for removal remain higher than desired (Keane and Parsons, 2010). Some fire treatments also increase ground fuel loads by causing blister rust–killed snags to fall, although such downed wood may be beneficial to whitebark pine regeneration by providing shelter supporting the establishment of seedlings (Keane and Parsons, 2010). In any case, returning fire to fire-suppressed whitebark pine forests is not simple, and its efficacy in restoration remains unknown. (Continued) Prepublication Copy 55

Forest Health and Biotechnology: Possibilities and Considerations BOX 3-1 Continued Ash Containment and management strategies related to emerald ash borer (EAB) have focused on prevention of further dispersal and diminishing insect pressure through insecticides and biological con- trol (Poland and McCullough, 2006). Evaluation of the effectiveness of EAB containment strategies (selective removals, quarantine, and insecticide treatments) has shown that they fail to prevent the dispersal of EAB (Mercader et al., 2011, 2016; McCullough and Mercader, 2012). Selective removal does not prevent dispersal once infestation is detected in a given tree because EAB has already dis- persed to uninfested hosts. Quarantines have also proven quite disappointing for preventing dispersal, partially for the same reason that selective removals are ineffective because of human agency. While quarantines slowed the movement of infested nursery stock and dispersal, the movement of infested saw logs and firewood continued. EAB hitchhiking on vehicles and trains has been documented in the United States and in Russia where green ash is widely planted as a street tree. Hitchhiking, rather than transport of infested wood, may be a major dispersal method between widely separated cities along the interstate highways in the United States (Prasad et al., 2010); this may explain the appearance of EAB in Boulder, Colorado, in 2016, more than 880 km distant from the nearest infestation in Omaha, Nebraska. With regard to insecticide treatments, no naturally occurring microbial insecticide (e.g., Ba- cillus thuringiensis) has proven effective in killing adult beetles via aerial application or in a forest setting (McCullough et al., 2015). Biocontrol efforts targeting EAB started in 2007 in Michigan. Researchers released three EAB par- asitoid species from China, the egg parasitoid Oobius agrili, the larval endoparasitoid Tetrastichus planipennisi, and the larval ectoparasitoid Spathius agrili (Federal Register, 2007; Bauer et al., 2015). T. planipennisi can effectively control EAB attacking ash saplings and young stump sprouts (Duan et al., 2017). Once the tree develops thick bark, the ovipositor is too short to reach the EAB larvae (Abell et al., 2012; Duan et al., 2017). Although studies show successful establishment of these parasitoids in some areas, the range of S. agrili (Hymenoptera:Braconidae) is limited by its lack of cold tolerance (Duan et al., 2012). Other parasitoids (e.g., S. galinae) had been approved for release as of 2018; they were expected to perform well in colder climates and have ovipositors that can penetrate the thicker bark of older trees (Belokobylskij et al., 2012; Duan et al., 2014). Long-term monitoring years after the release and establishment of one or more of the introduced parasitoids reveals that EAB still persists after ash population collapse, maintaining very low population levels on ash saplings as small as 2 cm in stem diameter (Aubin et al., 2015). Poplar Several chemical control methods have been developed that are effective against S. musiva, in- cluding repeated application of the fungicide benomyl to control the spread of cankers in the field (Ostry, 1987; Liang et al., 2014). Various biological control mechanisms using bacteria (Gyenis et al., 2003) and fungi (Yang et al., 1994) have also shown some success in controlling S. musiva. These practices may help inhibit the spread of S. musiva in nursery operations, but the extent and frequency of treat- ment required makes them impractical in operational plantations (Ostry, 1987) or in wild populations. Site Management Practices Whether to contain a pest from spreading or as a strategy for long-term management, there are a number of site management practices that create conditions unconducive to a pest outbreak. Use of chemi- cals, such as pesticides and fungicides, are common practices in managed forests. However, reliance on chemical controls is generally not a long-term solution because of the long-recognized potential conse- quences of widespread pesticide use. These consequences may include evolution of resistance in the pest, nontarget impacts, substantial expense associated with repeated treatments, and public opposition to wide- spread use of potential toxins (Mack et al., 2000; Gould et al., 2018). 56 Prepublication Copy

Mitigating Threats to Forest Health As in eradication efforts, quarantines may be put in place to prevent the movement of contaminated wood, and infested or infected trees may be removed. For example, when the Asian longhorned beetle (Anoplophora glabripennis) was discovered infesting multiple tree species in urban parks and suburban neighborhoods (Haack et al., 2010) in New York in 1996 and in Chicago in 1998, survey, chipping and burning of infested trees, and quarantined movement of potential host species (including nursery stock) were all rapidly implemented. Uninfested host trees within specific distances from infested trees were treated with insecticide. However, despite successful eradication in several sites, repeated introductions of the beetle means that it remains a threat to U.S. forests (Haack et al., 2010). With regard to emerald ash borer (EAB), tree removal, quarantine, and insecticides have not been effective (see Box 3-1). Another site management practice to minimize the conditions that favor the onset of an outbreak is thinning. Silvicultural thinning of managed stands is often conducted to improve growth; this practice also promotes individual vigor, increasing tree defenses against insect pests and pathogens (Gottschalk, 1993; Maher et al., 2018). For example, mountain pine beetle (Dendroctonus ponderosae) is one of the native insect pest species predicted to expand its distributional range under climate change (Raffa et al., 2013). Outbreaks of this species are driven by drought and even-aged stands of mature trees. In this situation, thinning stands and removing infested trees may reduce the beetle population below the outbreak threshold. Thinning of the understory also removes fuel for wildfire, reducing the probability of tree mortality. Pruning, rather than thinning, can also be an option in silvicultural stands. For example, branch prun- ing of white pines can reduce the impact of white pine blister rust (Ostry et al., 2010; Schwandt et al., 2010). This approach is feasible only where white pine species are in silvicultural management. Pruning focuses on protecting existing trees and their genetic diversity but does not increase the genetic resistance of future progeny. Maintaining diverse forests and planting mixed stands where the site naturally supported multiple species is another management tool to promote ecosystem resistance to insect pests and pathogens. More diverse forests are subject to lower levels of herbivory by insects than are more homogeneous forests, and this effect increases with the taxonomic distances among trees and with the proportion of unaffected species (Jactel and Brockerhoff, 2007). This phenomenon is due to the dilution effect of the host species, which reduces both population growth and spread of the insect when the host tree is not abundant (Keesing et al., 2006, 2010). However, the dilution effect may not be as strong when generalist species (those that make use of multiple tree species) are involved (Jactel and Brokerhoff, 2007). A similar dynamic is hypothesized for disease transmission, which can be diluted with a decrease in abundance of the host species. Even in the case of generalist pathogens, such as Phytophthora ramorum, a decrease in virulence has been observed under diverse host conditions (Haas et al., 2011). Furthermore, diverse forests will likely experience lower stress from climate change–related drought because diverse stands have higher productivity and resilience to drought than monospecific and low-diversity stands (Rasche et al., 2013). In this case, reduced intraspe- cies competition and niche partitioning for resources such as nutrients, light, and water are likely the causes. Where forests are naturally less diverse, with one or two predominant species, options other than managing for overstory diversity will likely be more effective and appropriate. Breeding to Enhance Resistance Plants that are tolerant of insect pests and pathogens maintain productivity despite the presence of the damaging species. Plants that are genetically resistant maintain productivity by reducing the ability of the insects and pathogens to establish and cause stress (Leimu and Koricheva, 2006). Both tolerant and resistant plants have characteristics that allow persistence and growth despite the presence of damaging insects and pathogens and may be used in breeding programs designed to reduce vulnerability of tree populations (Sniezko and Koch, 2017; Woodcock et al., 2017). In this report, the committee uses the term resistance to include tolerance, as the two responses cannot always be easily distinguished in the field without further research. Prepublication Copy 57

Forest Health and Biotechnology: Possibilities and Considerations Genetic resistance confers lack of or reduced susceptibility to an array of threats, including insects and diseases (Telford et al., 2015; Sniezko and Koch, 2017; Woodcock et al., 2017; Showalter et al., 2018). The nonnative insect pest or pathogen may cause high mortality in the affected species in the forest, but genetic variation often allows some individuals to survive. Many forest tree species have at least some genetic resistance, even if at low frequencies, to most pathogens or insects (see Box 3-2; Lattanzio et al., 2006; Sniezko and Koch, 2017; Woodcock et al., 2017). The first priority in selective resistance breeding is to answer the following questions: 1. Is there genetic resistance in the host tree, and if so, what type and degree of resistance exists within a tree and its progenies? 2. What is the geographical distribution of trees having resistant phenotypes? 3. What is the frequency of the resistant phenotypes within host tree populations? Finding suitable parent trees can be difficult. Additionally, finding resistant parent trees does not mean all of the progeny from the parent trees will be resistant (Sniezko et al., 2014; Sniezko and Koch, 2017) and restoration plantings need to account for this. Resistance is a phenotype that usually results from a complex interaction of multiple genes across a multiplicity of environments. The simple, one-gene, Mendelian, dom- inant pest resistance cases occupy much of the literature because such systems are tractable and can be studied within the time and funding limits imposed on academic research. However, as in crop species, in many cases this form of resistance may not be durable in forest trees (McDonald and Linde, 2002; Kinloch et al., 2004; Palloix et al., 2009); rather, durable resistance may only be possible with polygenic genetic mechanisms (see American chestnut case in Box 3-2). Evaluating the durability of resistance within indi- viduals and across generations (Mundt, 2014; Sniezko and Koch, 2017) will also be paramount because trees will be on the landscape for decades to centuries. Effectively applying selective-breeding programs requires that these rare cases of resistance be identified and propagated in greenhouses or seed orchards and intercrossed to generate progeny with polygenic resistance for deployment in restoration and reforestation programs (Sniezko and Koch, 2017; Woodcock et al., 2017). In the case of nonnative pathogens or insects, a greater understanding of controls on pest population dynamics in the home range of the pathogen or insect would provide a valuable perspective on what type of resistance might have a high likelihood of success. For example, in Asia, what resistance is present in the native white pine species, where presumably the pines and the white pine blister rust fungus have co- evolved? It would be helpful to know if similar resistance exists (at even low frequency) in North American white pines. In addition, in some areas of Asia, white pine blister rust has become problematic (La, 2009; Zhang et al., 2010). Knowing the cause of that change—perhaps due to new land management practices, evolution of greater virulence in the rust, or changing climate—would also be useful information. Further study from a global perspective to understand coevolved systems and how they can be disrupted will help design strategies to restore species (including how to use any biotech option) and help refine models to increase understanding of the potential long-term efficacy of resistance and its impacts over the landscape. Another way to introduce genetic resistance into a susceptible tree species is to hybridize the susceptible species with a related resistant tree species. After hybridization, the offspring are backcrossed with different trees of the susceptible tree species to maintain genetic diversity. In theory, repeated backcrossing will result in resistant trees with genomes that are almost entirely consistent with those of the susceptible parent species, with the exception of the regions containing alleles that confer resistance to the insect pest or pathogen (Wood- cock et al., 2017). In practice, this result is rarely achieved without intensive monitoring with DNA markers and large backcross population sizes. Backcross breeding is most effective for introgression of resistance when resistance is due to one or two dominant factors. Even in the simple case of only one or two factors, if one or both of these factors are recessive, the breeding strategy must include selfing or intercrossing alternat- ing with backcrossing. Backcross breeding strategies may be greatly accelerated using marker-aided selection or genomic selection, particularly when the goal is to capture multiple resistance factors from the nonnative species. A good example of a backcross breeding program is the one undertaken to introduce blight resistance into the American chestnut from the Chinese chestnut (see Box 3-2). 58 Prepublication Copy

Mitigating Threats to Forest Health BOX 3-2 Progress in Resistance Breeding in Case Study Species American Chestnut Initial attempts to breed chestnut with resistance to chestnut blight began in the 1920s but ended unsuc- cessfully in the 1960s (Steiner et al., 2017). A renewed effort began in earnest in the 1980s with the formation of The American Chestnut Foundation (TACF) and the American Chestnut Cooperators’ Foundation (ACCF) (Griffin et al., 2006). The ACCF has used breeding within the C. dentata species to complement use of hypovir- ulence (Jacobs et al., 2012). TACF pursued a hybrid and backcross method to incorporate resistance from the Chinese chestnut (Castanea mollissima) into the American chestnut (see Figure 3-2). This approach was initiated after it became clear that relatively little genetic resistance exists in native populations of American chestnut. The Chinese chestnut has, on average, moderate to good resistance to the blight fungus, but indi- vidual trees may have some susceptibility (Huang et al., 1996). Early crosses of the Chinese chestnut to the American chestnut showed that some F1 hybrids were resistant (Burnham, 1988; Anagnostakis, 2012). Based on this observation, it was assumed that the resistant hybrid phenotype was due to a small number of domi- nant genes. If that were true, then repeatedly backcrossing the hybrids to American chestnuts and selecting for resistance would ultimately result in a resistant chestnut with a high-percent of American chestnut ancestry. After several cycles of backcrossing, the resistant progeny populations could be intercrossed and selection continued for resistance as well as other American chestnut traits such as tree form and rapid growth. In this way, genotypes that were essentially American chestnut in phenotype but carried resistance to blight could be created. As of 2018, this program was still ongoing, with an objective of imparting the resistance from the Chinese chestnut while trying to capture the growth, adaptability, and other characteristics of the American chestnut. However, the degree of resistance from selection has been disappointing after over 30 years of backcrossing and intercrossing for a number of reasons, including the difficulty of phenotype evaluation and the resulting lack of information on the underlying genetic architecture of resistance. The first releases from TACF intercrossed populations in 2007 had American chestnut growth rate and form for the most part, but the degree of blight resistance needed for sustainable survival in natural forest areas had not yet been achieved as of the time the committee was writing its report (Steiner et al., 2017). The most advanced backcross hybrids are descendants of a small number of hybrids of American and Chinese chestnuts, particularly the Clapper hybrid (Clapper, 1963), the Graves hybrid (Graves, 1942) and a third hy- brid, “Nanking” (Diller et al., 1964). More than 17,000 descendants of these hybrids have been tested for resistance, site adaptation, growth, and form (Sisco, 2004; Hebard, 2006). Seed orchards have been estab- lished in Virginia and Pennsylvania to increase numbers of nuts needed to implement large-scale forest trials. A continuing strategy includes retaining only those trees with sufficient blight resistance and a timber-type growth form. Genomic selection, a strategy based on the association of phenotypic performance with genome- wide patterns of DNA polymorphisms, is also in progress. Blight-resistant chestnuts reintroduced in southern Appalachian regions will also need to have resistance to Phytophthora cinnamomi, the agent of ink disease. Recent work has demonstrated resistance in TACF populations descended from Chinese and American chestnuts and in some of the hybrid progeny of the European chestnut (C. sativa) by the Japanese chestnut (C. crenata) (Santos et al., 2015; Westbrook et al., 2018). At the time the committee wrote its report, resistance screening of young seedlings was under way and showed promise (Jeffers et al., 2012; Steiner et al., 2017) and could be combined in the future with blight resistance through breeding (Steiner et al., 2017). Whitebark Pine A selective breeding program for whitebark pine with resistance to white pine blister rust has been ongo- ing since the 1990s in the Oregon and Washington portions of the species’ range. Cones are collected from candidate parents identified in the field, and seedlings grown from those cones are infected with the disease at about age 2 or 3 using an inoculation system previously developed to identify resistance in other white pine species (see Figure 3-3). Seedling families are assessed for up to 5 years for the type and degree of re- sistance, and this information is used to rate the parent’s resistance (Sniezko et al., 2011, 2018). (Continued) Prepublication Copy 59

Forest Health and Biotechnology: Possibilities and Considerations BOX 3-2 Continued FIGURE 3-2 Overview of the theoretical expectations upon which the American Chestnut Foundation backcross breeding program was initially based. NOTE: The illustrated “recovery” of the native American chestnut with each round of backcrossing is based on theoretical expectations that are rarely achieved in practice unless DNA marker- assisted selection is used in every generation. SOURCE: Westbrook, 2017. The first seedling inoculation trials started in 2002; additional trials have been undertaken when seed from new parent tree selections becomes available. Through 2018, the progeny of 1,225 parent trees had been tested for rust resistance for the nine seed zones in Oregon and Washington. Of these 1,225 parent trees, preliminary resistance ratings were available for 1,002 trees. The data from the seedling trials suggest that 394 of these trees have levels of resistance that may be useful in restoration efforts. However, the fre- quency of resistance varies geographically (e.g., by breeding zone or management unit, see Figure 3-4), which adds logistical complications to the resistance discovery and deployment process. In one of these zones, only 2 of 28 tested parent trees have even marginally useable degrees of resistance, while in another seed zone, 93 of 106 trees have useable degrees of resistance. The degree of resistance in some populations of whitebark pine in the Oregon and Washington portions of the species range is high enough that land man- agers can collect seed from the highly rated parent trees to use immediately in restoration, without waiting the decade or more to establish orchards, then produce and distribute seed. (Continued) 60 Prepublication Copy

Mitigating Threats to Forest Health BOX 3-2 Continued FIGURE 3-3 Testing whitebark pine for resistance to white pine blister rust at USDA Forest Service’s Dorena Genetic Resource Center. NOTES: Two-year old seedling progeny of different parent trees are inoculated with the pathogen and evaluated for up to 5 years. Note the large difference in survival among seedling families (each in a separate 10-tree row plot); for example, in the far left row all seedlings from one parent tree are dead or dying (red) whereas the next row to the right has seedlings from another parent tree that are alive (green). SOURCE: R. Sniezko. Resistance breeding programs for white pine blister rust are also under way in the interior western part of the species range and in western Canada (Sniezko et al., 2011, 2018). This concerted effort will provide a good genetic base of resistant trees, permitting a restoration effort for each seed zone that initially contains a minimum number of resistant parent trees. The mechanism of resistance is still not well understood, but prog- eny tests suggest that the trait is polygenic, as is the case in other species of white pine (Kinloch and Dupper, 2002). Field trials (under way at the time the committee wrote its report) will more fully define the level of expected survival in resistant progeny in areas of varying rust hazard and environmental conditions. So, at least for whitebark pine (in at least some seed zones), selective breeding offers an efficient method to develop resistant seedlings for restoration. The restoration plantings will need to be followed to examine durability of the genetic resistance in whitebark pine as well as stability of resistance in different environments. One of the challenges of using native resistance is the need to protect the resistant parent trees from other sources of mortality while the next generation of seed trees is maturing. A number of the blister rust– resistant parents have already been lost to fires or to attack by mountain pine beetle (Dendroctonus ponder- osae). The semiochemical verbenone (an antiaggregation pheromone) has been used to protect individual trees from mountain pine beetle attacks (Perkins et al., 2015) and can be particularly useful to protect the resistant parent trees used for seed collections for the future restoration efforts. Verbenone is not 100 percent effective, and it needs to be applied each year as conditions warrant. Other chemicals, carbaryl and pyrethroid insecticides have also been registered to help protect trees from mountain pine beetle (Hastings et al., 2001; Fettig et al., 2013). As with white pine blister rust, genetic resistance to mountain pine beetle has been found (Six et al., 2018). Resistance of pine species to outbreaks of mountain pine bark beetle depends upon several factors including resin responses and secondary chemistry (Huber et al., 2004; Franceschi et al., 2005; Raffa et al., 2008). These resistance factors are generalized against many insects and pathogens, but natural selection has likely shaped their form and strength in forests that have experienced strong bark beetle pressure over millennia. However, Raffa et al. (2013) have indicated that the “typical” mechanisms of resistance to mountain pine beetle found in lodgepole and ponderosa pine (high resin production and induced defenses) are poorly developed in whitebark pine. This circumstance might be expected for a naïve host tree that has not had strong evolutionary pressure to develop costly defenses (Cudmore et al., 2010). Indeed, the lower overall resistance of naïve hosts that have had little to no exposure to bark beetle—including lodgepole and jack pine in areas where the beetle is expanding its populations (northern British Columbia and Alberta) and high-elevation whitebark pine—has been well docu- mented (Cudmore et al., 2010; Raffa et al., 2013; Bentz et al., 2015). (Continued) Prepublication Copy 61

Forest Health and Biotechnology: Possibilities and Considerations BOX 3-2 Continued FIGURE 3-4 Variation in genetic resistance (percent of seedlings with stem symptoms ~15 months after inoculation) to white pine blister rust in whitebark pine among different geographic populations from throughout the range of the species from a seedling inoculation trial initiated in 2007 at Dorena Genetic Resource Center. NOTES: The number of parents tested using half-sib families in each seed source mean is indicated above the bar. The 18 Oregon and Washington sources (first 18 bars from the left) represent individual National Forest, National Park, and Confeder- ated Tribes of Warm Springs boundaries from which the seedlots were collected. The right-most three bars refer to seedlots from California, Idaho, Montana, and British Columbia. Populations range from highly susceptible (blue, >60% of the progeny showing early stem symptoms) to moderately resistant (red, families showing <60% of the progeny with early stem symptoms). SOURCE: Sniezko et al., 2018. Likewise, the conventional wisdom that asserts that faster growing pines are more resistant to bark beetle may not hold with whitebark pine. In past outbreaks, whitebark pines that survived were slower growing than those that were killed (Margoles, 2011). This slow growth, along with evidence that whitebark pine may not have the capacity to produce strong defensive responses even when healthy (Raffa et al., 2013), indicates that prescriptive thinning to release the host from competition may fail to reduce mortality. However, resistance may still prove to be a powerful tool, although it may take a different form. Strong resistance to bark beetle has been described wherein trees escape attack, not through the production of strong resin or chemical defenses, but rather the opposite: by producing greatly reduced chemical profiles that interfere with beetle recognition or attraction to hosts. Mature whitebark pine surviving a recent outbreak have been found to be genetically distinct from beetle-susceptible trees (Six et al., 2018). Whether the putative genetic resistance to mountain pine beetle proves to be durable is uncertain (as is resistance to blister rust) and will need further study and confirmation from the field over time. Natural selection may be acting quickly to enhance adaptation to changing conditions. High selection pressure that results in strong natural selection for beetle resistance and drought tolerance would be particularly valuable in the vast inaccessible areas of whitebark pine’s range that not are amenable to active restoration. The same challenges exist in breeding and outplanting trees resistant to bark beetle as exist for resistance to blister rust, given whitebark pine’s long maturation period. Also, whitebark pine with resistance to blister rust remains susceptible to beetles, and whitebark pines with beetle resistance are typically susceptible to blister rust. An integrated approach will be needed to look for correlates of resistance to the two threats to include in breeding programs for the restoration of this tree. (Continued) 62 Prepublication Copy

Mitigating Threats to Forest Health BOX 3-2 Continued Ash All of the ash species native to North America have some susceptibility to the EAB, with the widely dis- tributed green ash and white ash being very susceptible. Monitoring forests for individual trees with intact canopies after most of the ash in the monitored plot have died from EAB was initiated a few years after EAB was detected in Michigan in 2002 (Knight et al., 2012). A small number of green and white ash trees (<1 percent) survive EAB attack many years longer than conspecifics in the same stand. These “lingering ash” show evidence of less severe EAB infestation, often accompanied by vigorous wound healing, and maintain a healthy crown for years after local conspecifics have died (Knight et al., 2012, 2013; Koch et al., 2015). Replicated studies reveal reproducible quantitative differences in defensive responses to EAB larvae between lingering ash and susceptible ash genotypes (Koch et al., 2015). Crosses between two lingering ash parents produce progeny with greater larval-killing response than either parent (see Figure 3-5). This result suggests that the parents have different partial resistance responses that have a genetic basis. These progeny can form the basis of a breeding program for “stacking” or pyramiding the allelic variants at multiple loci that may be responsible for the variety of partial-resistance phenotypes. Alternatively, the best progeny can be grafted and planted in seed orchards to enable natural intercrossing for the production of seed for restoration efforts. This approach requires a monitoring, breeding, and pheno- typing program. Monitoring is needed to identify more lingering ash from different areas of adaptation, to maintain genetic diversity. Breeding is needed to stack up the genetic factors that contribute to the resistance phenotypes. Finally, continued phenotypic screening of grafted clones and progeny of newly identified linger- ing ash verifies that the resistance phenotypes have a genetic basis. The advantage of the selective breeding approach, in this case, is that the basis of the resistance is polygenic. Plant pests and pathogens do not overcome polygenic resistance, as quickly as monogenic resistance (Parlevliet and Zadoks, 1977; Carson and Carson, 1989; Simmonds, 1991; Tuzun, 2001; Mundt, 2014). The work on finding additional resistant ash selections continues, as does the breeding. Field tests will be needed to determine the efficacy and durability of the resistance from the selective-breeding programs. The time line for restoration with genetically resistant ash will depend on the search for additional selections and the results in the field trials. FIGURE 3-5 Larval-killing response in the F1 progeny of two lingering ash parents. NOTES: Pies indicate responses of the parents (PE-L38 and PE-L41) and of individual progeny. Colors indicate, by tree, the proportion of host-killed larvae (brown), early instar larvae (L1-L2, green), and late instar larvae (L3–L4, blue) 8 weeks after egg application. Parent evaluation was done in replicated tests in previous years. SOURCE: Jennifer Koch, unpublished data. Backcross breeding to closely related resistant species is not an option for most of the North American Fraxinus, because these species are genetically incompatible with the Asian Fraxinus species that exhibit resistance to EAB. Black ash, a very susceptible North American riparian species, is compatible with the more resistant Asian Fraxinus, but a selective-breeding program, while possible, had not been undertaken as of 2018. If such a program did exist, then the use of transcriptome-based markers to identify quantitative trait loci regions would be feasible, because the whole genome sequence and deep transcriptome sequencing resources exist for Fraxinus species (Lane et al., 2016; Sollars et al., 2017). (Continued) Prepublication Copy 63

Forest Health and Biotechnology: Possibilities and Considerations BOX 3-2 Continued Poplar There have been numerous studies on the genetics of susceptibility and resistance of Populus species and hybrids to Sphaerulina musiva infection. An early study with a controlled cross suggested that eastern cottonwood (P. deltoides) carries a recessive allele that confers resistance and that interspecific hybrids be- tween eastern cottonwood and black cottonwood (P. trichocarpa) show intermediate resistance (Newcombe and Ostry, 2001). Similarly, a survey using a greenhouse assay of wild accessions collected in a hybrid zone in Alberta showed that susceptibility to stem cankers was highest in balsam poplar (P. balsamifera), lowest in eastern cottonwood, and intermediate in hybrids (LeBoldus et al., 2013). Furthermore, there was minimal interact ion between host genotype and fungal strain in susceptibility of hybrid cultivars, suggesting that re- sistance mechanisms could be enhanced by breeding (LeBoldus et al., 2009). Based on microscopic obser- vations of stem infections, it appears that one of the main mechanisms of canker resistance is the formation of necrophylactic periderm around the point of entry, inhibiting spread of the fungus (Qin and LeBoldus, 2014). Interestingly, because the fungus has necrotrophic characteristics, resistance mechanisms do not involve a typical hypersensitive response, whereby the host limits fungal spread through coordinated cell death around the point of infection. On the contrary, if the hypersensitive response is activated by the infection, this can actually enhance susceptibility to the fungus (Liang et al., 2014; Qin and LeBoldus, 2014). There is also some evidence that the chemical composition of the leaf cuticle can inhibit infection by S. musiva (Gonzales-Vigil et al., 2017). Furthermore, leaf and stem infection can be enhanced by stressful conditions such as ozone ex- posure (Woodbury et al., 1994) or drought (Maxwell et al., 1997). Using Biotechnology to Enhance Resistance Another way to generate resistant trees is through the use of biotechnology. Biotechnological research to introduce or modify traits in trees has been explored in a wide range of economically and ecologically important tree species throughout the world. Appendix C contains reports on biotech tree species at all stages of research and development from 1987 through 2018. Often the reports cover establishment of initial proof-of-concept transformation and regeneration systems, which demonstrate bacterial resistance genes from donor species. In species where this system is robust, the appendix includes reports demonstrating the incorporation of genes conferring various traits in the target species. These traits include insect and fungal resistance, early flowering, phytoremediation, tolerance to metal toxicity, herbicide tolerance, improvement of wood quality, changes to lignin content, and tolerance to drought, frost, and salt. The primary approach used has been transgenesis via Agrobacterium tumefaciens; however, the table includes reports describing biotechnological approaches such as RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR) (see Box 3-3). For a more comprehensive treatment of the developments in the field of tree biotechnology, see Chang et al. (2018). The first application for a field test of a biotech tree in the United States was submitted to USDA in 1989 for a poplar modified for glyphosate tolerance (Fillatti et al., 1987). As of 2018, about 700 permits have been issued by USDA-APHIS. However, only two tree species modified using biotechnology had been grown outside of field trials in the United States in that time.1 The first tree species to reach this stage was papaya (Carica papaya). Varieties with resistance to papaya ring spot virus incorporated via transgen- esis have been grown in Hawaiian orchards since 1998. The second was apple (Malus × domestica) in which RNAi has been used to suppress the expression of polyphenol oxidase genes, resulting in fruit flesh that does not brown when peeled or cut. Nonbrowning apples became available to U.S. consumers in 2017. 1 A variety of plum has been modified via Agrobacterium-mediated transgenesis to have resistance to plum pox virus, and this variety has met U.S. government regulatory requirements. However, it is not grown commercially as of 2018. 64 Prepublication Copy

Mitigating Threats to Forest Health BOX 3-3 Biotechnological Approaches Humans have been directly modifying DNA since 1973 (Cohen et al., 1973), and continuous technological advances have improved the efficiency and precision of biotechnology. More than four decades later, there are many biotechnology tools available to manipulate the DNA of almost any organism, including trees. The following is a brief description of the most commonly used approaches. Mutagenesis Although the frequency of naturally occurring gene sequence polymorphisms is high in most forest trees, methods have been developed for increasing variation by inducing mutations in the DNA sequence (direct mutagenesis). Many kinds of specific gene mutations have been induced in genes of forest trees. Mutations have been produced to knock out gene activity (loss-of-function mutations). Gain-of-function mutations can be produced through enhanced expression of specific genes. Partial reduction of specific gene function can also be induced by reducing the expression of the target gene (knock-down mutations). The technology for inducing mutations is diverse. Chemical mutagenesisa (Riyal, 2011) uses compounds such as ethyl methane sulfonate (EMS) to induce small mutations in DNA sequences. For example, Zayed et al. (2014) used EMS to induce mutations in the tropical tree species kelampayan (Neolamarckia cadamba) and petai belalang (Leucaena leucocephala) because they determined that these species have a relatively low genetic diversity. Chemical mutagenesis (EMS) of pollen from willow (Salix spp.) was shown to create new sequence variants, detected by high-throughput sequencing (Riyal, 2011). Plants frequently undergo somatic mutations, and these can generate novel genetic variation within a single individual. This kind of mutation is particularly important for trees with long lifespans such as oaks (Plomion et al., 2018) and clonal organisms such as aspen trees (Ally et al., 2010). These mutations can sometimes be adaptive and potentially useful for breeding, as in the case of a mutation in Eucalyptus melliodora that enhances resistance to herbivory in branches harboring the mutation (Padovan et al., 2013). Such mutations can be captured in breeding programs (if they enter the germ line) or propagated through rooted cuttings or by stem grafting to rapidly deploy resistance genes. Transgenesis Transgenesis involves inserting foreign genes or DNA fragments into cells of a target species to create a new gene sequence. The DNA sequence may be inserted into a target cell through a variety of techniques: 1. Biolistics is a technique that inserts DNA into plant cells by physical bombardment. Tiny metal beads coated with DNA are propelled at high velocity through the plant cell wall into the cells (Klein et al., 1987; Sanford et al., 1987). 2. Electric shock (electroporation) opens plant cell membranes, allowing DNA to enter cells (Fromm et al., 1985). 3. Microinjection (Neuhaus and Spangenberger, 1990) of needle-like silicon carbide fibers (whiskers) penetrate the cell wall to permit injection or uptake of DNA into cells (Kaeppler et al., 1990). 4. Agrobacterium-mediated transformation takes advantage of a genus of bacteria that infects plant cells and transfers long segments of DNA, which become integrated into the host plant genome. Scientists can splice genes of interest into the transferred DNA (Gohlke and Deeken, 2014). Regardless of the mechanism of delivery, once inside the cells, DNA may integrate into the genome and be expressed, thereby potentially introducing new traits into the recipient plant (Zupan and Zambryski, 1995). Transgenesis can also be used to induce mutations or alter the function of native genes. Loss-of-function mutations may occur when a sequence is inserted into a gene, and the gene function is thereby inactivated. Alternatively, gain-of-function mutations are created when an active sequence element, such as a promoter or enhancer, is inserted near a functional gene, causing an increase in the transcription of the target gene, which can lead to a novel or enhanced phenotype. Large numbers of transformation events are needed to screen for specific kinds of mutations in specific genes. Fortunately, large numbers of trees can be outplanted and maintained as a field archive, so that mutations can be expressed for mature traits in addition to juvenile ones (Busov et al., 2005a). (Continued) Prepublication Copy 65

Forest Health and Biotechnology: Possibilities and Considerations BOX 3-3 Continued Cisgenesis Cisgenesis is similar to transgenesis, but the inserted genes or DNA fragments are from an organism that is sexually compatible with the recipient organism. That is, the incorporation of the DNA into the target cell could possibly be accomplished with selective breeding, but biotechnology tools allow for the direct introduc- tion of the related DNA into the cell to achieve expression of the desired genetic trait. RNA Interference RNA interference is a molecular strategy common to all higher organisms for defense against parasites and pathogens, and regulation of native gene expression. Aspects of this natural process can be engineered to shut off (“silence”) specific genes in the parasite, insect, pathogen, or host plant by incorporating a small piece of the gene sequence in a configuration that results in the production of double-stranded RNA; this in turn activates the “dicer” complex, which degrades transcripts that match the gene fragment (Tang et al., 2003). Genome Editing Genome editing is a genetic modification process that makes specific and targeted changes to an organ- ism’s DNA. The four main classes of this approach are meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR) nuclease system. The CRISPR system generates fewer “off-target” changes and thus has become the favored approach among researchers (Iyer et al., 2018). The CRISPR toolbox is rapidly expanding as re- searchers develop innovative methods to manipulate this system (Wang et al., 2016), which has opened the door to using genome editing to introduce robust disease resistance genes into plants (Langner et al., 2018). Genome editing by CRISPR has significant potential for introducing specific changes in the genes of forest trees (Tsai and Xue, 2015; Elorriaga et al., 2018). For example, CRISPR editing in Chinese white poplar (Populus tomentosa) has been reported for a phytoene desaturase (PDS) gene, giving rise to albino pheno- types (Fan et al., 2015). CRISPR technology may make it possible to create homozygous biallelic DNA se- quence changes (Gantz and Bier, 2015), which would eliminate the need to intercross modified trees to ensure that the edited gene was in a homozygous state. This is a significant advantage for trees, which typically have long generation times and poor tolerance for inbreeding. Synthetic DNA Synthetic DNA refers to genes produced in the laboratory that are not based on any naturally occurring DNA sequences but that may have functional properties or utility for genetic engineering. They are different from naturally occurring genes in that they may be made or found to have unique metabolic functions. Syn- thetic genes are different from genes that are artificially synthesized but are based on known genes from any living microbe, plant, or animal. At the time the committee wrote its report, no truly synthetic genes had been introduced into a forest tree. In the future, novel synthetic genes could be of value for forest health, particularly for generating highly specific resistance to attacks by insect pests and pathogens if and when natural re- sistance genes are overcome by newly evolved variants of pests or pathogens. The committee is aware of the possibility of the eventual creation of such novel synthetic genes; however, the relevance to this report is purely hypothetical. aIn the U.S. regulatory system, chemical mutagenesis is not a regulated process. To use biotechnology to confer resistance to a forest health threat, the first step would be to identify the gene that would be targeted for modification, introduction, or silencing. If a gene target is not already in hand, then a gene discovery process would be required. This step has traditionally been hindered in trees due to the characteristics that make them difficult as experimental organisms: large size, long generation time, potentially weak correlations between seedling and adult phenotypes, and (in the case of conifers) immense genomes. Purification of high-quality genomic DNA from forest trees requires modest modifica- tions of standard procedures used for extracting DNA from plants due to high phenolic content, large fragile 66 Prepublication Copy

Mitigating Threats to Forest Health cells, or highly lignified tissue, such as stems or inner bark. The sheer size of many tree genomes presents a less tractable problem. Conifers have some of the largest genomes ever sequenced. The pine genomes can exceed 20,000 megabase pair (Mbp) per haploid genome (Zimin et al., 2014); sugar pine (Pinus lamberti- ana) has a genome size of 31,000 Mbp (Stevens et al., 2016). In comparison, Arabidopsis has only 135 Mbp and rice has 420 Mbp per haploid genome. Huge genome sizes and high content of repetitive DNA present difficulties in DNA sequencing and in genome assembly after sequencing. Another problem results from the sequence diversity of forest tree genomes. Forest trees have high levels of heterozygosity due to their large population sizes and outcrossing breeding systems (Williams et al., 1999; Remington and O’Malley, 2000). There are no inbred lines of forest trees and few, if any, haploid individuals. Consequently, sequencing a tree genome is made more difficult because, even for an individual tree, there are two diverse haploid genomes that are being sequenced at the same time, creating a challenge for sequence assembly. In sequencing and assembling the loblolly pine (Pinus taeda) genome, direct se- quencing of DNA from haploid megagametophyte seed storage tissue avoided this difficulty (Zimin et al., 2014). Unfortunately, the seed storage tissues of angiosperm (broad-leaved) forest trees (such as ash) are diploid or even polyploid, and so this strategy is not available. Genome sequences can provide substantial insights into organismal evolution, but their applicability in biotechnological approaches requires functional characterization of the components of the genome, including transcribed sequences (genes) and regulatory elements. One of the most significant methods for learning about the function of tree genomes is comparative sequence analysis, which reveals homologous sequences in dif- ferent genomes that, in turn, implies similar functions. Proof of function of homologs requires further testing, such as evidence of transcription and translation, purification of a functional product, or genetic complemen- tation. Here again, forest trees are at a severe disadvantage. Many advances in functional genomics have come from work on the genetic model plant Arabidopsis thaliana and herbaceous crops such as maize. Many tree species have structural and developmental differences that either required adaptation of methodology or pre- cluded application altogether. Furthermore, many characteristics common to trees (e.g., dormancy, wood for- mation, and obligate outcrossing) have few or no analogs in annual crop species. Recent technological developments have mitigated some of these shortcomings such that dramatic progress has been made in understanding the organization, structure, and function of tree genomes, thereby facilitating potential biotech modifications aimed at pressing problems in forest health. These developments include large-scale quantitative trait locus (QTL) or genome-wide association studies coupled with in-depth analyses of transcriptional and metabolic responses to insect or pathogen attack. Additionally, genome ed- iting can produce a “meiotic drive” (a kind of gene drive) function that converts a heterozygous individual to a homozygous one at one or more loci of interest, eliminating a generation of selfing to produce homo- zygous trees in one generation (see Box 3-4). The second phase of deployment in using biotechnology to modify phenotypes is production of trees containing the desired gene sequence. Transgenesis and genome editing require a transformation and tissue culture protocol in which the desired modification can be introduced into a single cell (usually in callus culture), and whole plants are generated from the transformed cell by regeneration of roots and shoots from disorganized callus tissue (organogenesis) (Birch, 1997). Many species of trees remain recalcitrant to the process of cell culture and regeneration. Even when regeneration is possible, the regeneration of a plant from a single cell may not produce an individual that has the desired genetic change in every cell. In well- studied plant species that are amenable to this process, embryos can be produced through somatic embryo- genesis, a process where the manipulated cell or cells originate from a totipotent embryo and then are in- duced to make more embryos (Hakman and Von Arnold, 1985; Suprasanna et al., 2005). Regeneration can be stepwise and sequential, where shoots are induced first and rooting is induced subsequently through organogenesis. This process is complex and must be customized not only at the species level but often for individual cultivars within a species (Busov et al., 2005a). The third phase is field testing. The case study species demonstrate varying degrees of progress with regard to the application of biotechnology to mitigat- ing forest health threats (see Box 3-5). Prepublication Copy 67

Forest Health and Biotechnology: Possibilities and Considerations BOX 3-4 Status of Gene-Drive Feasibility in Trees A gene drive is a system of biased inheritance in which the ability of a genetic element to pass from a parent to its offspring through sexual reproduction is enhanced, resulting in a preferential increase of a specific genotype that determines a specific phenotype in a population (NASEM, 2016). It can occur in nature (e.g., in mosquitoes and mice), and as of 2018, scientists were studying this process and others to develop engi- neered gene drives in various organisms. Introducing gene drives into an organism’s population may be of interest to reduce disease (e.g., to reduce the ability of mosquitoes to carry or transmit infectious diseases) or to control nonnative species. In trees, gene drives might be of interest to ensure the passage of resistance to a disease or insect from a modified tree on to the next generation. However, as of 2018, research into gene drives was still nascent, and much remained to be learned about the processes and possible impacts before employing their use outside the laboratory. Trees are not good can- didates for gene drive research because of their long generation times (NASEM, 2016). The insect pests that affect trees would be better subjects for research because of their short generation times; however, the potential effects on forest health from the modification of insects was outside the committee’s statement of task. BOX 3-5 Progress in Using Biotechnology to Confer Resistance to Case Study Species American Chestnut Building on work begun in the 1990s, the genes, transfer vectors, and technology for using biotechnology in the American chestnut has been developed (Merkle et al., 1991; Polin et al., 2006; Andrade et al., 2009; Barakat et al., 2009, 2012; Jabr, 2014; Newhouse et al., 2014a,b; Powell, 2014). The most promising candi- date gene for genetic resistance to chestnut blight was a wheat gene encoding the enzyme oxalate oxidase (OxO) (Polin et al., 2006; Welch et al., 2007). Oxalic acid (C2H2O4) is generated by the blight fungus during infection. The acid environment weakens plant cell walls, enabling other fungal enzymes to degrade the wall and the cell membranes, killing the cell (Dutton and Evans, 1996; Welch et al. 2007). In plants naturally pos- sessing an OxO gene, oxalate oxidase catalyzes the degradation of oxalic acid by converting it to carbon dioxide and hydrogen peroxide. The protein encoded by the OxO gene from wheat is effective against oxalic acid in tissues of the American chestnut and shows no evidence of toxicity to the host plant. Transgenesis using Agrobacterium-based vectors have successfully transferred OxO genes into the American chestnut (Andrade et al., 2009; Zhang et al., 2011). A fast and accurate in vitro leaf assay was developed to detect OxO activity in the leaves of transformed and regenerated plants early in plant develop- ment (Newhouse et al., 2014a). Investigators have now shown that transfer and expression of a wheat OxO gene in the American chestnut confers a degree of resistance equivalent to or greater than that found in the Chinese chestnut (Zhang et al., 2013; Newhouse et al. 2014a). The OxO-transformed plants derived from the original transformant are named “Darling” American chestnut trees after Herbert Darling, former president of the New York Chapter of TACF. The Darling 58 genotype has been characterized for blight resistance, growth and form, nutritional composition, lack of toxicity to the host plant, stability of blight tolerance, nontarget inter- actions, and lack of effects on target organisms (see Figure 3-6; Newhouse, 2018). Whitebark Pine No effort to date has focused on utilizing biotechnology to impart genetic resistance in whitebark pine. The large genome size of conifers, limited information on the genome of whitebark pine and candidate re- sistant genes, and biotechnology tools available suggest using biotechnology would take perhaps a decade or much longer to produce a resistant tree. With resistance breeding in whitebark pine imparting a more im- mediate and cost-effective solution, there may be little need to explore this option for whitebark pine, unless (a) the resistance(s) identified in the selective breeding method prove to be not durable (e.g., evolution of virulence in the pathogen), (b) some populations (seed zones) of whitebark pine have little or no inherent resistance and using seedlots from other seed zones is deemed not suitable for the environmental conditions to which they would be moved, or (c) additional unique types of resistance were identified (not found in current whitebark pine) and deemed necessary to complement the current resistance from breeding to help ensure that the trees stay resistant into the future. Even if biotechnology is used, the seed production would likely be through the development of seed orchards, which would add at least two decades to the production of resistant seed. (Continued) 68 Prepublication Copy

Mitigating Threats to Forest Health BOX 3-5 Continued FIGURE 3-6 Wild-type American chestnut seedlings (left), Darling transgenic American chestnut seedlings (middle), and Chinese chestnut seedlings (right). SOURCE: Bill Powell, SUNY-ESF. Available at http://parrottlab.uga.edu/SIV B/HTML/Darling%2054%20American%20chestnut%20small%20stem%20assay%209-11-15%20SUNY-ESF%20D SC_0160.html. A potential advantage of biotechnology for whitebark pine is that it may be possible to develop seedlots with a higher frequency of resistant seedlings than in the initial generation of parent trees now being used. However, it may be simpler and more efficient to plan to plant extra seedlings from the selective-breeding resistance program (perhaps only 20 to 40 percent of seedlings from any current resistant parent tree will be rust resistant). Perhaps the real potential (future) utility of biotechnology for developing resistant whitebark pine would be if new types of genetic resistance (of a durable nature), not found in whitebark pine were identified and transferred into whitebark pine. Although researchers are cautiously optimistic about the durability of ge- netic resistant to the rust that is developed through the selective breeding program, knowledge of a backup option, provided through biotechnology, would be useful to have. Significant research and trials of this material would likely take one to several decades for any future deployment. Ash Protocols for applying biotechnology to North American ash have not been established. Micropropagation techniques are reported for European common ash (F. excelsior), narrow leafed ash (F. angustifolia, native to southern Europe, northwest Africa, and southwest Asia), and green, white, and black ash (Hammatt, 1994; Schoenweiss and Meier-Dinkel, 2005; Capuana, 2012; Beasley and Pijut, 2013; Li et al., 2014; Lee and Pijut, 2017). Successful Agrobacterium-mediated transformation is reported for green, white, and pumpkin ash (F. profunda) (Du and Pijut, 2009; Stevens and Pijut, 2014; Palla and Pijut, 2015). The lack of reports on the successful insertion and stable expression of a gene or genes effective against the emerald ash borer (EAB) are due, in part, to a lack of vetted genes. As of 2018, there was not an active program in the United States to develop reproducible and stable transformation systems for Fraxinus, although a stable micropropagation pro- tocol suitable for gene transfer applications had recently been developed for both F. pennsylvanica (Li et al., 2014) and F. Americana (Merkle et al., 2017). Pijut and her colleagues mention studies under way for insertion of the Cry8Da protein of the bacterium B. thuringiensis into F. pennsylvanica and other Fraxinus, but there are no reports in the literature or in the patent databases of success, as defined by integration of the gene into the genome, expression of the gene, and efficacy of the gene product against EAB. Toxicity studies have shown some efficacy of Cry8Da formulations against EAB adults when the preparation is sprayed on leaves (Bauer and Londoño, 2010). Toxicity tests on larvae await the development of an artificial diet that results in normal growth. The only alternative is to trans- form a susceptible ash, prove that the gene is expressed, grow the transformant to a suitable size, bud (Continued) Prepublication Copy 69

Forest Health and Biotechnology: Possibilities and Considerations BOX 3-5 Continued graft to save the genotype and enable replicated studies, tape EAB eggs on the saplings, and track the fate of each hatched egg when the stem is dissected 8 weeks after taping (Koch et al., 2015). This testing process requires 8–10 years, assuming that the transformation system requires 4–5 years to develop and deploy. This transformation system estimate is less than the time actually required for development of a reliable micro- propagation and successful transformation system for American chestnut (Merkle et al., 1991; Carraway and Merkle, 1997; Andrade et al., 2009; Kong et al., 2014). There are no reports on efforts to use genome-editing techniques in Fraxinus due to insufficient knowledge of the gene expression networks involved in defensive responses. Recent studies have featured deep sequencing transcriptome analyses, proteomes, and metabolomes in phloem or leaf tissues in other tree species (Hamanishi et al., 2015; Fu et al., 2016; Wu et al., 2016; Nguyen et al., 2017), but there are few studies in angiosperm trees that capture the transcriptome, proteome, and metabolome associated with re- sponse to stem-boring insects. Comparison of the defensive enzymes and metabolites in the phloem tissues of Manchurian ash (F. mandshurica) and black ash reveal few qualitative differences in either constitutive or induced phenolics, despite the resistance of Manchurian ash and the susceptibility of black ash (Whitehill et al., 2012). Studies of EAB larvae fed on Manchurian, green, and white ash reveal similar levels of detoxifica- tion enzymes in the insect regardless of the species food source, even though the phloem phenolic profiles of Manchurian ash differ considerably from the green ash and white ash (Cipollini et al., 2011). The most informative study, focused on a more detailed investigation of uninfested Manchurian ash and black ash, showed higher levels of peroxidases, lignin polymerization, and quinone generation in Manchurian ash (Rigsby et al., 2016). Based on these studies, peroxidases, lipoxygenases, chitinases, polyphenol oxidases, and other defense-related enzymes are expected in the proteome profiles of uninfested Manchurian ash. The defensive enzyme and metabolite profiles of infested and uninfested Manchurian ash and black ash subjected to water stress indicated nonqualitative changes in metabolites in both species, including a higher accumula- tion of pinoresinol A in infested Manchurian ash only when both species were not subjected to water stress (Chakraborty et al., 2014). The transcriptomes, proteomes, and metabolomes of the North American Fraxinus remain uninvestigated in controlled experiments, wherein genotypes of the same species grafted and grown at the same time are compared, infested versus uninfested. Once phenotyping strategies and statistical de- signs have sufficient power to identify differences in defensive responses directly attributable to EAB attack, genome-editing approaches could be possible provided that micropropagation and transformation techniques for North American Fraxinus are improved at the same time. Poplar There have been several notable developments in the genomics of the Sphaerulina-Populus pathosys- tem. The genomes of both the main host (P. deltoides) and the fungus have been sequenced by the U.S. Department of Energy’s Joint Genome Institute (Dhillon et al., 2015). The fungal genome sequencing project also included the closely related Sphaerulina populicola, which does not cause cankers and has a broader host range within Populus. Both genomes are approximately 30 Mbp and contain about 10,000 genes in highly collinear and syntenic order. Genes that are specific to S. musiva are enriched for wood-degrading enzymes. Furthermore, S. musiva contains a co-regulated gene cluster that was apparently horizontally transferred from Penicillium fungus. This cluster is induced upon exposure to Populus wood and encodes genes with phyto- toxic, antifungal, and antibacterial activities (Dhillon et al., 2015). There have also been a number of functional genomics studies of S. musiva in recent years that have provided additional insights into the infection process. For example, an RNAseq study revealed a potential fungal elicitor (SMEcp2) that is expressed during the infection process. Treatment of stems of susceptible individuals with the isolated protein caused lesions, suggesting that this elicitor induces necrosis in the host (Dunnell, 2016). Bioinformatic analysis of the genome sequences revealed another secreted peptide (RALF27) that is present in both S. populicola and S. musiva, but absent in other closely related fungi. In fact, the closest match in public databases is to the RALF27 gene of P. deltoides, suggesting another case of horizontal transfer, but this time from host to fungus, potentially enhancing virulence (Thynne et al., 2017). (Continued) 70 Prepublication Copy

Mitigating Threats to Forest Health BOX 3-5 Continued On the host side, an RNAseq analysis in hybrid Populus demonstrated up-regulation of genes involved in oxidation-reduction, protein fate, secondary metabolism, and defense-related gene products, all of which is consistent with expectations. However, genes related to the hypersensitive response were also up-regulated in susceptible host genotypes, supporting the hypothesis that susceptibility to this necrotrophic pathogen may be enhanced by triggering programmed cell death in the host (Liang et al., 2014). Similar results were ob- served in P. deltoides, for which the jasmonate and ethylene signaling pathways were induced in response to infection with S. musiva, along with genes involved in lignin biosynthesis and cell wall modification (Foster et al., 2015). One of the reasons that Populus has become a favored model organism is the ease with which it is maintained and propagated in tissue culture and from vegetative cuttings. This facilitates the development and large-scale implementation of biotechnology-based methods of tree improvement (Busov et al., 2005a). Early efforts focused on developing spontaneous mutants in tissue culture with enhanced resistance to S. musiva, but these results translated poorly from the greenhouse to the field (Ostry and Ward, 2003). Targeted attempts to enhance host resistance with Agrobacterium-mediated transformation have been more success- ful. Overexpression of the antimicrobial peptides AMP1.2 and ESF12 enhanced resistance of a hybrid Populus clone to S. musiva based on a leaf disk assay (Liang et al., 2002). Similarly, overexpression of the OxO gene from wheat in hybrid Populus increased resistance to leaf infection by S. musiva (Liang et al., 2001). This recapitulates the success with this gene against the chestnut blight, Cryphonectria parasitica. However, unlike the chestnut results, the Populus transgenics have not yet been tested in the field, and efficacy against can- kers has not yet been demonstrated. A method developed to transform S. musiva using Agrobacterium (Foster et al., 2014) holds great promise to enhance understanding of the infection process and possibly to provide control measures aimed at the fungus using gene drives (Gantz et al., 2015). For example, following the sequencing of the black cottonwood (Populus trichocarpa) genome (Tuskan et al., 2006), it became possible to tag genes by insertional mutagen- esis to identify the sites of insertion and to isolate the genes that activated to produce a phenotype (Busov et al., 2005b). Three types of insertional mutagenesis have been carried out for poplars. Mutagenesis by inser- tion of T-DNA from Agrobacterium (Fladung et al., 2004; Busov et al., 2005b), insertion of a maize Ac trans- posable element (Howe et al., 1991), and insertion of reporter genes that are activated when inserted near a promoter or an expression enhancer (enhancer traps) (Groover et al., 2004). Enhancer traps are sequences containing a low-expression reporter gene that is activated if inserted near the promoter of an active gene, giving rise to a reporter gene phenotype (Springer, 2000). TIME LINES AND COSTS OF DIFFERENT MANAGEMENT OPTIONS FOR FOREST HEALTH The speed and cost of approaches to mitigate threats to forest health can vary widely. They are variable not only by approach (e.g., biological control, breeding, or biotechnology) but also by the state of knowledge about the target tree and the target pest (and its potential predator). Speed and Cost of Biological Control The speed and cost of biological control efforts vary depending on the biology of the target pest and the availability of biocontrol agents. A best-case scenario might be represented by the ash whitefly (Siphoninus phillyreae Haliday), an exotic insect from Eurasia and Africa that caused extensive defoliation of urban ornamental trees in California between 1988 and 1991 (Pickett et al., 1996). A wasp parasitoid, Encarsia inaron Walker, was imported from Italy and Israel, reared and tested under confined conditions, and then released into 43 counties in California by 1992. This resulted in nearly total control of the ash whitefly within the first year, with populations in Riverside, California, being reduced by a factor of 10,000 (Bellows et al. 1992). The total investment for this program was estimated at $1.2 million, and the cost savings were estimated to be between $220 million and $300 million, based primarily on the retail cost of removing and replacing urban trees (Pickett et al., 1996). This case was simpler than average for several reasons. First, the insect was restricted to California, and so federal regulations about interstate movement Prepublication Copy 71

Forest Health and Biotechnology: Possibilities and Considerations were not a factor, and only one state regulatory agency and USDA were involved. Second, the parasitoid was readily available and highly specific, so the risk assessment was simplified. A more typical biological control effort could be expected to cost between $2 million and $5 million and take 5–10 years to complete (Dr. Mark Hoddle, University of California, Riverside, personal communication, August 20, 2018). Other biocontrol efforts may not be successful, as has been the case for EAB (see Box 3-1). Additionally, the introduction of nonnative predators to control nonnative pests can often take years, first to identify the appropriate predators and second to obtain regulatory approval for their use (Rose, 2018). Speed of Selective Resistance Breeding The time line for selective resistance breeding in forest trees is dependent on several factors. In the best- case scenario for selective breeding, the infrastructure for a breeding program already exists, tree breeding expertise exists, and the biology of the tree species and the insect or pathogen is known. As discussed above (see section “Breeding to Enhance Resistance”), the first steps in selective breeding are to determine whether there is genetic resistance within the affected species, the frequency of resistance over its range, and type and degree of resistance available (i.e., is it immediately useable or will breeding be required?). Determining the frequency and distribution of resistance, where it exists, may take several years or decades. Seed collection and testing of seedlings from hundreds or thousands of parent trees may be nec- essary. Those steps must then be followed with the development of breeding or orchard populations that have useful types and degrees of genetic resistance and sufficient genetic diversity to use for restoration (e.g., Sniezko et al., 2012; Dudley et al., 2017). Two of the fastest evolving selective-breeding programs in forest trees have been development of resistance to the soilborne pathogen Phytophthora lateralis in Port-Orford-cedar (Chamaecyparis lawsoniana) on the West Coast (Sniezko et al., 2012) and development of resistance to the fungal disease Fusarium oxysporum in the Hawaiian koa tree (Acacia koa; Dudley et al., 2017). However, these two op- erational resistance programs only progressed rapidly once the basic data about resistance had been col- lected. In Port-Orford-cedar, the initial assessment erroneously concluded that there was no resistance (Han- sen et al., 1989). In the koa, there was initial uncertainty as to the causative agent of mortality. The operational program for resistance in Port-Orford-cedar started in 1996 (after a significant research period), and the first orchard seed was released by 2003. The operational koa wilt resistance program started in 2003; the first orchard seed was not available until over a decade later. Even though programs for these two species were producing resistant seed as of 2018, the work was not complete. Seed was available for only some breeding zones, and the number of resistant parent trees in some orchards is too low to ensure that genetic diversity is preserved. By contrast, the program to develop white pine blister rust resistance in sugar pine has taken longer to develop because of lower degrees of resistance and the longer time to reproductive maturity in sugar pine. The sugar pine resistance breeding program has continued for 50 years, with slow but steady progress (Sniezko et al., 2000; Kegley and Sniezko, 2004; McDonald et al., 2004; Kinloch et al., 2008, 2012). The case of whitebark pine with resistance to white pine blister rust demonstrates the variation in the time it takes from resistant parent identification to deployment of resistant seedlings based on geography and parent genetics (Sniezko et al., 2007, 2011). Six restoration plantings were established in Crater Lake National Park from 2009 to 2016 (see Figure 3-7), using seedlings from some of the most resistant parents from the park. Seedling testing of progeny of Crater Lake parent trees was started in 2004, making the time from first testing to the first restoration planting only 5 years. However, seed orchards are also planned for Oregon and Washington seed zones, and in this case it may take from 10 to 20 years before resistant seed from these orchards is available. Thus, the overall time to begin restoration efforts using selective resistance breeding with whitebark pine can vary from as little as 5 years to several decades or more. The highly sporadic nature of good cone crops can also slow progress in resistance testing of candidate trees. Even in the case of Crater Lake, additional parent trees are being evaluated to increase the genetic base of seedlots used for restoration. With enough funding, a good cone crop, and a good seed collection effort, hundreds or even thousands of whitebark pine parent trees could be evaluated in a short time. 72 Prepublication Copy

Mitigating Threats to Forest Health FIGURE 3-7 Restoration planting of whitebark pine (established 2009) at Crater Lake National Park. PHOTO CREDIT: R. Sniezko. The backcross breeding program for the American chestnut which occurred over six generations took 35 years, a relatively short amount of time for several generations of tree breeding (see Figure 3-2). How- ever, the program did not include the establishment of resistant seed orchards, the incorporation of genetic diversity in resistant seeds, or the development of resistant populations of chestnut for different geographic areas. Relative Speed and Cost of Biotechnological Approaches in Trees One of the commonly cited advantages of biotechnological approaches to create resistant genotypes of trees is the speed with which they can be deployed. Selective breeding and backcrossing are slow pro- cesses in some tree species (such as sugar pine) because the low initial degree of resistance and the long juvenile periods of most trees translate to long generation times and therefore very slow breeding cycles (Harfouche et al., 2012; Isik et al., 2015). Furthermore, there is often a poor correlation between traits measured in juvenile trees compared to those in mature trees, which necessitates expensive field testing over multiple years for each breeding cohort (White et al., 2007). Consequently, most forest breeding pro- grams have only progressed through a few of generations, leading to modest genetic gains compared to annual commodity crops such as maize and wheat (Isik et al., 2015). Biotechnology has been promoted as a means to accelerate the domestication of forest trees by shortening the breeding cycle (e.g., through early flowering; Martín-Trillo and Martínez-Zapater, 2002; Flachowsky et al., 2009), using marker-aided selec- tion (Harfouche et al., 2012; Isik et al., 2015), or bypassing breeding entirely by manipulating DNA (Merkle and Dean, 2000; Harfouche et al., 2011). The actual speed of biotechnological approaches depends on a number of practical factors that can potentially limit implementation. In the area of marker-aided selection, it is becoming increasingly clear that the efficacy of this approach is limited by the complex genetic architecture of quantitative traits. Ge- nome-wide association studies have clearly demonstrated that complex traits are polygenic, that is, con- trolled by hundreds or even thousands of loci, each of which has small genetic effects, and complex epistatic interactions (Boyle et al., 2017). This complexity means that alleles that control traits in one population are often not effective at predicting phenotypes in an unrelated population, thus requiring expensive and time- consuming marker discovery and model training in each subpopulation (Resende et al., 2012). Although this problem should be diminished for some disease resistance traits with simpler genomic architecture (e.g., for cases of major gene resistance), these types of resistance are expected to be less durable than quantitative resistance based on multiple unlinked loci (McDonald and Linde, 2002), so applications are limited. Prepublication Copy 73

Forest Health and Biotechnology: Possibilities and Considerations The situation could be different when using biotechnology to make individual genetic modifications to produce dramatic phenotypic changes. For example, introduction of the crystalline endotoxins derived from Bacillus thuringiensis (Bt) can confer complete resistance to feeding by Lepidoptera (moths and but- terflies), even in host species that are normally highly susceptible to such damage (Shelton et al., 2002). A wide variety of Bt toxins are already available (de Maagd et al., 2003), and existing toxins can be modified using mutagenesis to enhance efficacy against a particular insect once introduced into the plant via transgen- esis (de Maagd et al., 1999). Similar examples exist for pathogen resistance as well, such as the introduction of the oxalate oxidase gene (see Box 3-5), which shows broad efficacy against fungal pathogens such as the Cryphonectria blight in American chestnut (Zhang et al., 2013) and Septoria leaf spots in Populus (Liang et al., 2001). Nevertheless, the relative speed with which biotechnology solutions can be imple- mented depends on a number of factors, and significant impediments at each stage of development could substantially slow the process. As reviewed above (see section “Using Biotechnology to Enhance Resistance”), the first step to make use of biotechnology to introduce genetic resistance is gene identification. Although the gene discovery process has become remarkably more efficient with advances in genome sequencing technology, this step could take a number of years. For example, in the case of the emerald ash borer, there was no genome or transcriptome sequence available for the insect or host in the early stages of the epidemic, and it is time- consuming and difficult to measure ash tree resistance, which requires infestation of trees that are 2–3 years old (Koch et al., 2015). The second step is producing trees with the desired gene sequence. Under the best-case scenario, represented by the model transformation clones in Populus, it takes 4–8 months to produce transformed plants that could be transplanted to pots (Busov et al., 2010). At least another several months would then be required to vegetatively propagate enough material for a field trial. In other species for which transfor- mation systems are not readily available, the process of regenerating a plant from somatic embryos to a seedling growing under ordinary conditions in a greenhouse can take more than 1 year. If the introduced gene is present in the germ line, progeny of the transformed plant will also have the inserted gene. In many tree species it can take 5–10 years or more until flowers are produced and the gene can be passed on to the next generation. The third step is field testing. The length of this phase would depend on the growth rates of the trees and the life history of the insect pest or pathogen. In some cases, resistance is best evaluated in adult trees, which requires many years. For example, both Septoria and Cryphonectria cankers take years to develop, though effective in vitro assays have been developed for both diseases (LeBoldus et al., 2010; Newhouse et al., 2014b). Furthermore, multiple field trials over a large geographic area are desirable, especially in cases where significant genotype × environment interactions occur for host susceptibility. Given the ex- pense and difficulty of performing field trials, testing would typically begin on a limited basis to demon- strate efficacy before scaling up to larger and more widespread trials. This slow rollout would add years to the process. As a result, the full process would take over a decade. Estimating costs of the application of biotechnology for forest health is difficult because it requires estimation of processes and products that have not yet been developed or information that is proprietary and not available. The cost of gene identification or developing a new DNA transfer system is undefined because in some cases the efforts could be unsuccessful; therefore, the project could have high costs with no results. Producing trees with the desired gene sequence through clonal propagation can be expensive. However, when it comes to a comparison of costs between breeding and biotechnology, the costs may be similar or at least similarly variable. With regard to the identification of a trait of interest, for the biotech tree the expense is in gene discovery and integrating the desired change into the tree’s DNA; for breeding, the costs are related to screening and testing to find resistance. For both approaches, the costs can vary widely depending on the biology of the tree and the pest, the state of knowledge about the tree and pest biology, and the robustness of the biotech or breeding program associated with the species of interest. When it comes to the next step of clonal propagation, the costs are likely to be similar between the two approaches. 74 Prepublication Copy

Mitigating Threats to Forest Health The major difference in costs between the two would be those associated with any regulatory approval that a biotech tree may need to obtain; a selectively bred or hybrid tree does not have to go through a regulatory process in the United States. CONCLUSIONS AND RECOMMENDATIONS Management to mitigate damage to forests from insects or pathogens takes significant time and re- sources. With regard to nonnative insects and pathogens, the first line of defense is preventing their intro- duction. When introduced pests have become established or native pests are expanding their range or in- creasing in virulence, there are a number of management options that may be employed, including taking no action. Chemical or biological control can control pests in some cases, but these approaches are often not acceptable to the public, effective, or timely. The development and actual deployment of genetic re- sistance, whether via breeding or biotechnology, will usually take decades from the initial research phase to even the beginning of the restoration plantings. However, given the repeated introduction of nonnative pests and the likelihood of continued abiotic stress from climate change, incorporating genetic resistance may be the effective strategy for the long term. Conclusion: Substantial literature supports the need for sustained investment in prevention and eradication as the most cost-effective and lowest impact approaches for managing introduction of nonnative insect pests and pathogens. Economic analysis has found that the United States could save billions of dollars in avoided impacts from nonnative pests by increasing its efforts to prevent the entry of nonnative pests. Inspection, quarantine, and treatment of imported materials can facilitate the interception of insect pests and pathogens prior to their potential escape and establishment. Recommendation: Investment in effective prevention and eradication approaches should be the first line of defense against nonnative species in efforts to maintain forest health. Conclusion: Any single management practice alone is not likely to be effective at combatting major pest outbreaks. Site management practices—such as pesticide use, thinning, reintroduction of fire, and removal of infested trees—can minimize conditions that favor a pest outbreak. Biological control agents can suppress insect pest populations or mitigate the effects of a fungal pathogen. However, experience with the American chestnut, whitebark pine, ash, and poplar indicates that these practices will be insufficient to curtail the loss of affected tree species. Recommendation: Management for forest health should make use of multiple practices in combina- tion to combat threats to forest health. Conclusion: A variety of biotech and nonbiotech approaches have been and will be developed to ad- dress insect pest and pathogen threats. The time line for use of these tools in management activities for forest trees and forest health will depend on a number of factors, but the biology of the species involved (both tree and insect or pathogen) and the environments in which the tree species exist will have a major influence on effective mitigation. The time line for using approaches to mitigate forest health such as biological control, breeding, or biotechnology vary by the state of knowledge about the target tree and the target pest. The availability of natural enemies, the size of the tree genome, and the environment will also affect the deployment of miti- gation tools. Prepublication Copy 75

Forest Health and Biotechnology: Possibilities and Considerations Conclusion: Many tree species have some degree of resistance to particular native and nonnative pests that may be harnessed to combat infestations and epidemics. It is often possible to find resistance to damaging insects and diseases in the field and use it to develop resistant trees for restoration planting. However, this outcome depends on the resources to find resistant trees and established breeding programs to develop resistant seedlings. This strategy has been successfully deployed for blister rust resistance in whitebark pine and, at the time of the committee’s report, were also in use for ash against EAB. For whitebark pine, there are still no programs for drought tolerance or re- sistance to mountain pine beetle. Recommendation: Entities concerned about forest health should devote resources to identifying re- sistant trees within a population that have survived a pest outbreak. Research to understand the role of resistance in coevolved systems from the perspective of a global host–pest system, where the nonnative pathogen or insect originate, would help guide efforts in North America. Conclusion: Using biotechnology to introduce resistance to threats in forest trees has been hampered by the complexity of tree genomes, the genetic diversity in tree populations, and the lack of knowledge about genetic mechanisms that underlie important traits. However, recent technological develop- ments have improved functional genomic tools, facilitating the potential for biotechnology to help address forest health problems. At the time the committee was writing its report, there was insufficient knowledge about the funda- mental mechanisms involved in resistance to pests to efficiently identify genomic means to mitigate pest damage. Most tree genomes had not been sequenced, and there were still many unknowns about the under- lying nature of resistance, including its heritability and on whether it will be durable. Investigations in trees species are needed to uncover all forms of resistance, not just those due to easily discernible single major genes. Likewise, in using biotechnology, greater efforts are needed to understand what types of resistance or combinations of resistance are likely to be durable. Recommendation: More research should be conducted on the fundamental mechanisms involved in trees’ resistance to pests and adaptation to diverse environments, including a changing climate. Conclusion: The time it takes to identify resistance in an affected population, breed resistant seed- lings, and plant resistant seedlings in the field can vary from a few years to multiple decades, depend- ing on the species. Incorporating resistance via biotechnology into a tree species is also a lengthy process, the duration of which varies by species. The amount of natural genetic resistance in a population can vary by species or by the geographic distribution of a species, and the reproductive cycle of the tree will affect how quickly resistant offspring can be generated. When introducing resistance via biotechnology, the number of the genes involved in the expression of resistance will affect how long it takes to identify and incorporate resistance in biotech trees. Recommendation: Sufficient investment of time and resources should be made to successfully identify or introduce resistance into tree species threatened by insects and pathogens. REFERENCES Abell, K.J., J.J. Duan, L. Bauer, J.P. Lelito, and R.G. Van Driesche. 2012. The effect of bark thickness on host parti- tioning between Tetrastichus planipennisi (Hymen: Eulophidae) and Atanycolus spp. (Hymen: Braconidae), two parasitoids of emerald ash borer (Coleop: Buprestidae). Biological Control 63(3):320–325. 76 Prepublication Copy

Mitigating Threats to Forest Health Ally, D., K. Ritland, and S.P. Otto. 2010. Aging in a long-lived clonal tree. PLoS Biology 8:e1000454. Anagnostakis, S.L. 2012. Chestnut breeding in the United States for disease and insect resistance. Plant Disease 96(10):1392–1403. Anagnostakis, S.L. and B. Hillman. 1992. Evolution of the chestnut tree and its blight. Arnoldia 52(2):3–10. Andrade, G.M., C.J. Nairn, H.T. Le, and S.A. Merkle. 2009. Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation. Plant Cell Reports 28(9):1385–1397. Arno, S.F., and R.J. Hoff. 1989. Silvics of whitebark pine (Pinus albicaulis). Ogden, UT: U.S. Forest Service. Aubin, I., F. Cardou, K. Ryall, D. Kreutzweiser, and T. Scarr. 2015. Ash regeneration capacity after emerald ash borer (EAB) outbreaks: Some early results. The Forestry Chronicle 91(3):291–298. Barakat, A., D.S. Diloreto, Y. Zhang, C. Smith, K. Baier, W.A. Powell, N. Wheeler, R. Sederoff, and J.E. Carlson. 2009. Comparison of the transcriptomes of American chesnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) in response to chestnut blight infection. BMC Plant Biology 9:51. Barakat, A., M. Staton, C.-H. Cheng, J. Park, N.B.M. Yassin, S. Ficklin, C.-C. Yeh, F. Hebard, K. Baier, W. Powell, S.C. Schuster, N. Wheeler, A. Abbott, J.E. Carlson, and R. Sederoff. 2012. Chestnut resistance to the blight disease: Insights from transcriptome analysis. BMC Plant Biology 12:38. Bauer, L.S., J.J. Duan, J.R. Gould, and R.G. Van Driesche. 2015. Progress in the classical biological control of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) in North America. The Canadian Entomologist 147(3):300–317. Bauer, L.S., and D.K. Londoño. 2010. Effects of Bacillus thuringiensis SDS-502 on adult emerald ash borer. Pp. 74– 75 in Proceedings: 21st U.S. Department of Agriculture Interagency Research Forum on Invasive Species 2010. Newtown Square, PA: U.S. Forest Service. Beasley, R.R., and P.M. Pijut. 2013. Regeneration of plants from Fraxinus nigra Marsh. Hypocotyls. HortScience 48(7):887–890. Belokobylskij, S.A., G.I. Yurchenko, J.S. Strazanac, A. Zaldívar-Riverón, and V. Mastro. 2012. A new emerald ash borer (Coleoptera: Buprestidae) parasitoid species of Spathius nees (Hymenoptera: Braconidae: Doryctinae) from the Russian Far East and South Korea. Annals of the Entomological Society of America 105(2):165–178. Bellows, T.S., T.D. Paine, J.R. Gould, L.G. Bezark, J.C. Ball, W. Bentley, R.L. Coviello, J. Downer, P. Elam, D. Flaherty, P. Gouveia, C. Koehler, R.H. Molinar, N.V. O’Connell, E. Perry, and G. Vogel. 1992. Biological control of ash whitefly: A success in progress. California Agriculture 46(1):24–28. Bentz, B.J., C.K. Boone, and K.F. Raffa. 2015. Tree response and mountain pine beetle attack preference, reproduction and emergence timing in mixed whitebark and lodgepole pine stands. Agricultural and Forest Entomology 17(4):421–432. Birch, R.G. 1997. PLANT transformation: Problems and strategies for practical application. Annual Review of Plant Physiology and Plant Molecular Biology 48(1):297–326. Boland, G.J. 2004. Fungal viruses, hypovirluence, and biological control of Sclerotinia species. Canadian Journal of Plant Pathology 26(1):6–18. Boyle, E.A., Y.I. Li, and J.K. Pritchard. 2017. An expanded view of complex traits: From polygenic to omnigenic. Cell 169(7):1177–1186. Burgiel, S., G. Foote, M. Orellana, and A. Perrault. 2006. Invasive alien species and trade: Integrating prevention measures and international trade rules. Washington, DC: Center for International Environmental Law and Defend- ers of Wildlife. Available at https://cleantrade.typepad.com/clean_trade/files/iastraderpt0106.pdf. Accessed No- vember 8, 2018. Burnham, C.R. 1988. The restoration of the American chestnut. American Scientist 76(5):478–487. Busov, V.B., A.M. Brunner, R. Meilan, S. Filichkin, L. Ganio, S. Gandhi, and S.H. Strauss. 2005a. Genetic transfor- mation: A powerful tool for dissection of adaptive traits in trees. New Phytologist 167(1):9–18. Busov, V., M. Fladung, A. Groover, and S. Strauss. 2005b. Insertional mutagenesis in Populus: Relevance and feasi- bility. Tree Genetics & Genomes 1(4):135–142. Busov, V., S.H. Strauss, and G. Pilate. 2010. Transformation as a tool for genetic analysis of Populus. Pp. 113–133 in Genetics and Genomics of Populus, S. Jansson, R. Bhalerao, and A. Groover, eds. New York: Springer. Capuana, M. 2012. In vitro propagation of ash (Fraxinus excelsior L.) by somatic embryogenesis. Pp. 213–221 in Protocols for Micropropagation of Selected Economically-Important Horticultural Plants, M. Lambardi, E.A. Ozudogru, and S.M. Jain, eds. New York: Springer. Carraway, D.T., and S.A. Merkle. 1997. Plantlet regeneration from somatic embryos of American chestnut. Canadian Journal of Forest Research 27(11):1805–1812. Carson, S.D., and M.J. Carson. 1989. Breeding for resistance in forest trees—a quantitative genetic approach. Annual Review of Phytopathology 27(1):373–395. Prepublication Copy 77

Forest Health and Biotechnology: Possibilities and Considerations Chang, S., E.L. Mahon, H.A. MacKay, W.H. Rottmann, S.H. Strauss, P.M. Pijut, W.A. Powell, V. Coffey, H. Lu, S.D. Mansfield, and T.J. Jones. 2018. Genetic engineering of trees: Progress and new horizons. In Vitro Cellular & Developmental Biology-Plant 54(4):341–376. Chakraborty, S., J.G.A. Whitehill, A.L. Hill, S.O. Opiyo, D.O.N. Cipollini, D.A. Herms, and P. Bonello. 2014. Effects of water availability on emerald ash borer larval performance and phloem phenolics of Manchurian and black ash. Plant, Cell & Environment 37(4):1009–1021. Choi, G.H., A.L. Dawe, A. Churbanov, M.L. Smith, M.G. Milgroom, and D.L. Nuss. 2012. Molecular characterization of vegetative incompatibility genes that restrict hypovirus transmission in the chestnut blight fungus Cryphonectria parasitica. Genetics 190(1):113–127. Cipollini, D., Q. Wang, J.G.A. Whitehill, J.R. Powell, P. Bonello, and D.A. Herms. 2011. Distinguishing defensive characteristics in the phloem of ash species resistant and susceptible to emerald ash borer. Journal of Chemical Ecology 37(5):450–459. Clapper, R.B. 1963. A promising new forest-type chestnut tree. Journal of Forest 61(12):921–922. Cohen, S.N., A.C.Y. Chang, H. Boyer, and R.B. Helling. 1973. Construction of biologically functional bacterial plas- mids in vitro. Proceedings of the National Academy of Sciences of the United States of American 70(11):3240– 3244. Cortesi, P. and M.G. Milgroom. 1998. Genetics of vegetative incompatibility in Cryphonectria parasitica. Applied Environmental Microbiology 64(8):2988–2994. Cudmore, T.J., N.B. Björklund, A.L. Carroll, and B.S. Lindgren. 2010. Climate change and range expansion of an aggressive bark beetle: Evidence of higher beetle reproduction in naïve host tree populations. Journal of Applied Ecology 47(5):1036–1043. de Maagd, R.A., D. Bosch, and W. Stiekema. 1999. Bacillus thuringiensis toxin-mediated insect resistance in plants. Trends in Plant Science 4(1):9–13. de Maagd, R.A., A. Bravo, C. Berry, N. Crickmore, and H.E. Schnepf. 2003. Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annual Review of Genetics 37(1):409–433. Diller, J.D., R.B. Clapper, and R.A. Jaynes. 1964. Cooperative test plots produce some promising Chinese and hybrid chestnut trees. U.S. Forest Service Research Note NE-25. Upper Darby, PA: U.S. Forest Service. Dhillon, B., N. Feau, A.L. Aerts, S. Beauseigle, L. Bernier, A. Copeland, A. Foster, N. Gill, B. Henrissat, P. Herath, K.M. LaButti, A. Levasseu, E.A. Lindquist, E. Majoor, R.A. Ohm, J.L. Pangilinan, A. Pribowo, J.N. Saddler, M.L. Sakalidis, R.P. de Vries, I.V. Grigoriev, S.B. Goodwin, P. Tanguay, and R.C. Hamelin. 2015. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen. Proceedings of the National Academy of Sciences of the United States of America 112(11): 3451–3456. Du, N., and P.M. Pijut. 2009. Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration. Plant Cell Reports 28(6):915–923. Duan, J.J., L.S. Bauer, and R.G. Van Driesche. 2017. Emerald ash borer biocontrol in ash saplings: The potential for early stage recovery of North American ash trees. Forest Ecology and Management 394:64–72. Duan, J.J., G. Yurchenko, and R. Fuester. 2012. Occurrence of emerald ash borer Coleoptera: Buprestidae and biotic factors affecting its immature stages in the Russian Far East. Environmental Entomology 41(2):245–254. Duan, J.J., T.J. Watt, and K. Larson. 2014. Biology, life history, and laboratory rearing of Spathius galinae (Hymenoptera: Braconidae), a larval parasitoid of the invasive emerald ash borer (Coleoptera: Buprestidae). Journal of Economic Entomology 107(3):939–946. Dudley, N., T. Jones, R. James, R. Sniezko, J. Wright, C. Liang, P.F. Gugger, and P. Cannon. 2017. Applied genetic conservation of Hawaiian Acacia koa: An eco-regional approach. Pp. 78–91in Gene Conservation of Tree Spe- cies—Banking on the Future, Proceedings of a Workshop, R.A. Sniezko, G. Man, V. Hipkins, K. Woeste, D. Gwaze, J.T. Kliejunas, and B.A. McTeague, tech. cords. Portland, OR: U.S. Forest Service. Dunnell, K.L. 2016. Understanding Host-Pathogen Interactions in the Sphaerulina musiva-Populus Pathosystem. Ph.D. dissertation, Oregon State University. Dutton, M.V., and C.S. Evans. 1996. Oxalate production by fungi: Its role in pathogenicity and ecology in the soil environment. Canadian Journal of Microbiology 42(9):881–895. Elorriaga, E., A.L. Klocko, C. Ma, and S.H. Strauss. 2018. Variation in mutation spectra among CRISPR/Cas9 mutagenized poplars. Frontiers in Plant Science 9:594. Fan, D., T. Liu, C. Li, B. Jiao, S. Li, Y. Hou, and K. Luo. 2015. Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation. Scientific Reports 5:12217. Fettig, C.J., D.M. Grosman, and A.S. Munson. 2013. Advances in insecticide tools and tactics for protecting conifers from bark beetle attack in the western United States. Pp. 472–492 in Insecticides—Development of Safer and More Effective Technologies, S. Trdan, ed. Rijeka, Croatia: InTech. 78 Prepublication Copy

Mitigating Threats to Forest Health Fillatti, J.J., J. Sellmer, B. McCown, B. Haissig, and L. Comai. 1987. Agrobacterium mediated transformation and regeneration of Populus. Molecular and General Genetics 206(2):192–199. Finnoff, D., J.F. Shogren, B. Leung, and D. Lodge. 2007. Take a risk: Preferring prevention over control of biological invaders. Ecological Economics 62(2):216–222. Flachowsky, H., M.V. Hanke, A. Peil, S.H. Strauss, and M. Fladung. 2009. A review on transgenic approaches to accelerate breeding of woody plants: Review. Plant Breeding 128(3):217–226. Fladung, M., F. Deutsch, H. Hönicka, and S. Kumar. 2004. T‐DNA and transposon tagging in aspen. Plant Biology 6(1):5–11. Foster, A.J., M.J. Morency, A. Séguin, and P. Tanguay. 2014. Agrobacterium tumefaciens–mediated transformation for targeted disruption and over expression of genes in the poplar pathogen Sphaerulina musiva. Forest Pathol- ogy 44(3):233–241. Foster, A.J., G. Pelletier, P. Tanguay, and A. Séguin. 2015. Transcriptome analysis of poplar during leaf spot infection with Sphaerulina spp. PLoS ONE 10(9):e0138162. Franceschi, V.R., P. Krokene, E. Christensen, and T. Krekling. 2005. Anatomical and chemical defenses of conifer bark against bark beetles and other pests. New Phytologist 167(2):353–376. Fromm, M., L.P. Taylor, and V. Walbot. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proceedings of the National Academy of Sciences of the United States of America 82(17):5824– 5828. Fu, S., J. Shao, C. Zhou, and J.S. Hartung. 2016. Transcriptome analysis of sweet orange trees infected with “Candidatus Liberibacter asiaticus” and two strains of citrus tristeza virus. BMC Genomics 17(1):349. Gantz, V.M., and E. Bier. 2015. The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science 348:442–444. Gantz, V.M., N. Jasinskiene, O. Tatarenkova, A. Fazekas, V.M. Macias, E. Bier, and A.A. James. 2015. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Pro- ceedings of the National Academy of Sciences of the United States of America 112(49):E6736–E6743. GAO (U.S. Government Accountability Office). 2015. Aquatic Invasive Species: Additional Steps Could Help Measure Federal Progress in Achieving Strategic Goals. Available at: https://www.gao.gov/assets/680/673897.pdf. Ac- cessed June 18, 2018. Gohlke, J., and R. Deeken. 2014. Plant responses to Agrobacterium tumefaciens and crown gall development. Fron- tiers in Plant Science 5:155. Gonzales-Vigil, E., C.A. Hefer, M.E. von Loessl, J. La Mantia, and S.D. Mansfield. 2017. Exploiting natural variation to uncover an alkene biosynthetic enzyme in poplar. The Plant Cell 29(8):2000–2015. Gottschalk, K.W. 1993. Silvicultural Guidelines for Forest Stands Threatened by the Gypsy Moth. Radnor, PA: U.S. Forest Service. Gould, F., Z.S. Brown, and J. Kuzma. 2018. Wicked evolution: Can we address the sociobiological dilemma of pes- ticide resistance? Science 360(6390):728–732. Graves, A.H. 1942. Breeding work toward the development of a timber type of blight resistant chestnut. American Journal of Botany 29(8):622–626. Grente, J., and S. Sauret. 1969. L’hypovirulence exclusive, phénomene original en pathologie végétale. Comptes Ren- dus Hebdomadaire des Séances de l’Académie d’Agriculture de France. Série D 286:2347–2350. Grente, J., and S. Berthelay-Sauret. 1978. Biological control of chestnut blight in France. Pp. 30–34 in Proceedings of the American Chestnut Symposium, W.L. McDonald., F.C. Cech, J. Luchok, and C. Smith, eds. Morgantown: West Virginia University Press. Griffin, G.J. 2000. Blight control and restoration of the American chestnut. Journal of Forestry 98(2):22–27. Griffin, G.J., J.R. Elkins, D. McCurdy, and S.L. Griffin. 2006. Integrated use of resistance, hypovirulence, and forest management to control blight on American chestnut. Pp. 97–108 in Proceedings of Restoration of American Chestnut to Forest Lands, K.C. Steiner and J.E. Carlson, eds. Washington, DC: National Park Service. Groover, A., J.R. Fontana, G. Dupper, C. Ma, R. Martienssen, S. Strauss, and R. Meilan. 2004. Gene and enhancer trap tagging of vascular-expressed genes in poplar trees. Plant physiology 134(4):1742–1751. Gyenis, L., N.A. Anderson, and M.E. Ostry. 2003. Biological control of Septoria leaf spot disease of hybrid poplar in the field. Plant Disease 87(7):809–13. Haack, R.A., K.O. Britton, E.G. Brockerhoff, J.F. Cavey, L.J. Garrett, M. Kimberley, F. Lowenstein, A. Nuding, L.J. Olson, J. Turner, and K.N Vasilaky. 2014. Effectiveness of the International Phytosanitary Standard ISPM no. 15 on reducing wood borer infestation rates in wood packaging material entering the United States. PLoS ONE 9:e96611. Prepublication Copy 79

Forest Health and Biotechnology: Possibilities and Considerations Haack, R.A., F. Hérard, J. Sun, and J.J. Turgeon. 2010. Managing invasive populations of Asian longhorned beetle and citrus longhorned beetle: A worldwide perspective. Annual Review of Entomology 55:521–546. Haas, S.E., M.B. Hooten, D.M. Rizzo, and R.K. Meentemeyer. 2011. Forest species diversity reduces disease risk in a generalist plant pathogen invasion. Ecology Letters 14(11):1108–1116. Hakman, I., and S. Von Arnold. 1985. Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce). Journal of Plant Physiology 121(2):149–158. Hamanishi, E.T., G.L.H. Barchet, R. Dauwe, S.D. Mansfield, and M.M. Campbell. 2015. Poplar trees reconfigure the transcriptome and metabolome in response to drought in a genotype- and time-of-day-dependent manner. BMC Genomics 16(1):329. Hammatt, N. 1994. Shoot initiation in the leaflet axils of compound leaves from micropropagated shoots of juvenile and mature common ash (Fraxinus excelsior L.). Journal of Experimental Botany 45(6):871–875. Hansen, E.M., P.B. Hamm, and L.F. Roth. 1989. Testing Port-Orford-cedar for resistance to Phytophthora. Plant Disease 73(10):791–794. Harfouche, A., R. Meilan, and A. Altman. 2011. Tree genetic engineering and applications to sustainable forestry and biomass production. Trends in Biotechnology 29(1):9–17. Harfouche, A., R. Meilan, M. Kirst, M. Morgante, W. Boerjan, M. Sabatti, and G.S. Mugnozza. 2012. Accelerating the domestication of forest trees in a changing world. Trends in Plant Science 17(2):64–72. Hastings, F.L., E.H. Holsten, P.J. Shea, and R.A. Werner. 2001. Carbaryl: A review of its use against bark beetles in coniferous forests of North America. Environmental Entomology 30(5):803–810. Hebard, F.V. 2006. The backcross breeding program of the American Chestnut Foundation. Journal of The American Chestnut Foundation 19(2):55–77. Howe, G.T., S.H. Strauss, and B. Goldfarb. 1991. Insertion of the maize transposable element Ac into poplar. Pp. 283–294 in Woody Plant Biotechnology, M.R. Ahuja, ed. New York: Plenum. Huang, H., W.A. Carey, F. Dane, and J.D. Norton. 1996. Evaluation of Chinese chestnut cultivars for resistance to Cryphonectria parasitica. Plant Disease 80:45–47. Huber, D.P.W., S. Ralph, and J. Bohlmann. 2004. Genomic hardwiring and phenotypic plasticity of terpenoid-based defenses in conifers. Journal of Chemical Ecology 30(12):2399–2418. Isik, F., S. Kumar, P.J. Martínez-García, H. Iwata, and T. Yamamoto. 2015. Acceleration of forest and fruit tree domestication by genomic selection. Advances in Botanical Research 74:93–124. Iyer, V., K. Boroviak, M. Thomas, B. Doe, L. Riva, E. Ryder, and D.J. Adams. 2018. No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice. PLOS Genetics 14(7):e1007503. Jabr, F. March 1, 2014. A new generation of American chestnut trees may redefine America’s forests. Scientific American 310. Jacobs, D.F., H.J. Dalgleish, and C.D. Nelson. 2012. A conceptual framework for restoration of threatened plants: The effective model of American chestnut (Castanea dentata) reintroduction. New Phytologist 197(2):378–393. Jactel, H., and E.G. Brockerhoff. 2007. Tree diversity reduces herbivory by forest insects. Ecology Letters 10(9):835– 848. Jeffers, S.N., I.M. Meadows, J.B. James, and P.H. Sisco. 2012. Resistance to Phytophthora cinnamomi among seed- lings from backcross families of hybrid American chestnut. Pp. 194–195 in Proceedings of the Fourth Interna- tional Workshop on the Genetics of Host–Parasite Interactions in Forestry: Disease and Insect Resistance in Forest Trees, R.A. Sniezko, A.D. Yanchuk, J.T. Kliejunas, K.M. Palmieri, J.M. Alexander, and S.J. Frankel, tech. cords. Albany, CA: U.S. Forest Service. Kaeppler, H.F., W. Gu, D.A. Somers, H.W. Rines, and A.F. Cockburn. 1990. Silicon carbide fiber-mediated DNA delivery into plant cells. Plant Cell Reports 9(8):415–418. Kalaris, T., D. Fieselmann, R. Magarey, M. Colunga-Garcia, A. Roda, D. Hardie, N. Cogger, N. Hammond, P.T. Martin, and P. Whittle. 2014. The role of surveillance methods and technologies in plant biosecurity. Pp. 309– 337 in The Handbook of Plant Biosecurity. Dordrecht, The Netherlands: Springer. Keane, R.E., and R.A. Parsons. 2010. Management guide to ecosystem restoration treatments: Whitebark pine forests of the northern Rocky Mountains, U.S.A. Fort Collins, CO: U.S. Forest Service. Keesing, F., R.D. Holt, and R.S. Ostfeld. 2006. Effects of species diversity on disease risk. Ecology Letters 9(4):485- 498. Keesing, F., L.K. Belden, P. Daszak, A. Dobson, C.D. Harvell, R.D. Holt, P. Hudson, A. Jolles, K.E. Jones, C.E. Mitchell, S.S. Myers, T. Bogich, and R.S. Ostfeld. 2010. Impacts of biodiversity on the emergence and trans- mission of infectious diseases. Nature 468(7324):647–652. Kegley, A. and R.A. Sniezko. 2004. Variation in blister rust resistance among 226 Pinus monticola and 217 P. lambertiana seedling families in the Pacific Northwest. Pp. 209–226 in Breeding and Genetic Resources of 80 Prepublication Copy

Mitigating Threats to Forest Health Five-needle Pines: Growth, Adaptability, and Pest Resistance, R.A. Sniezko, S. Samman, S.E. Schlarbaum, and H.B. Kriebel, eds. Fort Collins, CO: U.S. Forest Service. Kenis, M., B.P. Hurley, A.E. Hajek, and M.J. Cock. 2017. Classical biological control of insect pests of trees: Facts and figures. Biological Invasions 19(11):3401–3417. Kinloch, B.B., Jr.,and G.E. Dupper. 2002. Genetic specificity in the white pine-blister rust pathosystem. Phytopathol- ogy 92(3):278–280. Kinloch, B.B., Jr., D. Burton, D.A. Davis, R.D. Westfall, J. Dunlap, and D. Vogler. 2012. Strong partial resistance to white pine blister rust in sugar pine. Pp. 80–91 in Proceedings of the Fourth International Workshop on the Genetics of Host–Parasite Interactions in Forestry: Disease and Insect Resistance in Forest Trees, R.A. Sniezko, A.D. Yanchuk, J.T. Kliejunas, K.M. Palmieri, J.M. Alexander, and S.J. Frankel, tech. cords. Albany, CA: U.S. Forest Service. Kinloch, B.B., Jr., D.A. Davis and D. Burton. 2008. Resistance and virulence interactions between two white pine species and blister rust in a 30-year field trial. Tree Genetics & Genomes 4(1):65–74. Kinloch, B.B., Jr., R.A. Sniezko, and G.E. Dupper. 2004. Virulence gene distribution and dynamics of the white pine blister rust pathogen in western North America. Phytopathology 94(7):751–758. Klein, T.M., E.D. Wolf, R. Wu, and J.C. Sanford. 1987. High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327(6117):70–73. Knight, K.S., J.P. Brown, and R.P. Long. 2013. Factors affecting the survival of ash Fraxinus spp. trees infested by emerald ash borer Agrilus planipennis. Biological Invasions 15(2):371–383. Knight, K.S., D. Herms, R. Plumb, E. Sawyer, D. Spalink, E. Pisarczyk, B. Wiggin, R. Kappler, E. Ziegler, and K. Menard. 2012. Dynamics of surviving ash (Fraxinus spp.) populations in areas long infested by emerald ash borer (Agrilus planipennis). Pp. 143–152 in Proceedings of the Fourth International Workshop on the Genetics of Host–Parasite Interactions in Forestry: Disease and Insect Resistance in Forest Trees, R.A. Sniezko, A.D. Yanchuk, J.T. Kliejunas, K.M. Palmieri, J.M. Alexander, and S.J. Frankel, tech. cords. Albany, CA: U.S. Forest Service. Koch, J.L., D.W. Carey, M.E. Mason, T.M. Poland, and K.S. Knight. 2015. Intraspecific variation in Fraxinus pennsylvanica responses to emerald ash borer (Agrilus planipennis). New Forests 46(5–6):995–1011. Kong, L., C.T. Holtz, C.J. Nairn, H. Houke, W.A. Powell, K. Baier, and S.A. Merkle. 2014. Application of airlift bioreactors to accelerate genetic transformation in American chestnut. Plant Cell, Tissue and Organ Culture 117(1):39–50. La, Y.-J. 2009. Korean successes in controlling blister rust of Korean pine. Pp. 1–9 in Breeding and Genetic Resources of Five-Needle Pines Conference, D. Noshad, E. Noh, J. King, and R. Sniezko, eds. Yangyang: Korea Forest Research Institute. Lane, T., T. Best, N. Zembower, J. Davitt, N. Henry, Y. Xu, J. Koch, H. Liang, J. McGraw, S. Schuster, D. Shim, M.V. Coggeshall, J.E. Carlson, and M.E. Staton. 2016. The green ash transcriptome and identification of genes responding to abiotic and biotic stresses. BMC Genomics 17:702. Langner, T., S. Kamoun, and K. Belhaj. 2018. CRISPR crops: Plant genome editing toward disease resistance. Annual Review of Phytopathology 56(1):479–512. Lattanzio, V., V.M. Lattanzio, and A. Cardinali. 2006. Role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects. Phytochemistry: Advances in Research 661(2):23–67. LeBoldus, J.M., P.V. Blenis, and B.R. Thomas. 2010. A method to induce stem cankers by inoculating nonwounded populus clones with Septoria musiva spore suspensions. Plant Disease 94(10):1238–1242. LeBoldus, J.M., P. Blenis, B.R. Thomas, N. Feau, and L. Bernier. 2009. Susceptibility of Populus balsamifera to Septoria musiva : A field study and greenhouse experiment. Plant Disease 93(11):1146–1150. LeBoldus, J.M., N. Isabel, K.D. Floate, P. Blenis, and B. R. Thomas. 2013. Testing the “hybrid susceptibility” and “phenological sink” hypotheses using the P. balsamifera–P. deltoides hybrid zone and Septoria leaf spot [Septoria musiva]. PLoS ONE 8(12):e84437. Lee, J.H., and P.M. Pijut. 2017. Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra. Plant Cell, Tissue and Organ Culture 130(2):335–343. Leimu, R., and J. Koricheva. 2006. A meta‐analysis of tradeoffs between plant tolerance and resistance to herbivores: Combining the evidence from ecological and agricultural studies. Oikos 112(1):1–9. Leung, B., M.R. Springborn, J.A. Turner, and E.G. Brockerhoff. 2014. Pathway-level risk analysis: The net present value of an invasive species policy in the US. Frontiers in Ecology and the Environment 12(5):273–279. Li, D., J. Zhang, and S.A. Merkle. 2014. Induction of green ash embryogenic cultures with potential for scalable somatic embryo production using suspension culture. Trees 28(1):253–262. Prepublication Copy 81

Forest Health and Biotechnology: Possibilities and Considerations Liang, H., C.M. Catranis, C.A. Maynard, and W.A. Powell. 2002. Enhanced resistance to the poplar fungal pathogen, Septoria musiva, in hybrid poplar clones transformed with genes encoding antimicrobial peptides. Biotechnol- ogy Letters 24(5):383–389. Liang, H., C.A. Maynard, R.D. Allen, and W.A. Powell. 2001. Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene. Plant Molecular Biology 45(6):619–629. Liang, H., M. Staton, Y. Xu, T. Xu, and J. Leboldus. 2014. Comparative expression analysis of resistant and suscep- tible Populus clones inoculated with Septoria musiva. Plant Science 223:69–78. Liebhold, A.M., E.G. Brockerhoff, S. Kalisz, M.A. Nuñez, D.A. Wardle, and M.J. Wingfield. 2017. Biological inva- sions in forest ecosystems. Biological Invasions 19(11):3437–3458. Liebhold, A.M., L. Berec, E.G. Brockerhoff, R.S. Epanchin-Niell, A. Hastings, D.A. Herms, J.M. Kean, D.G. McCullough, D.M. Suckling, P.C. Tobin, and T. Yamanaka. 2016. Eradication of invading insect populations: From concepts to applications. Annual Review of Entomology 61:335–352. Liu, Y.-C., M.L. Double, W.L. MacDonald, and M.G. Milgroom. 2002. Persistence of Cryphonectria hypoviruses after their release for biological control of chestnut blight in West Virginia forests. Forest Pathology 32(6):345– 356. Liu, Y.-C., and M.G. Milgroom. 1996. Correlation between hypovirus transmission and the number of vegetative incompatibility (vic) genes different among isolates from a natural population of Cryphonectria parasitica. Phytopathology 86(1):79–86. Lovett, G.M., M. Weiss, A.M. Liebhold, T.P. Holmes, B. Leung, K.F. Lambert, D.A. Orwig, F.T. Campbell, J. Rosen- thal, D.G. McCullough, R. Wildova, M.P. Ayres, C.D. Canham, D.R. Foster, S.L. LaDeau, and T. Weldy. 2016. Nonnative forest insects and pathogens in the United States: Impacts and policy options. Ecological Applications 26(5):1437–1455. Mack, R.N., D. Simberloff, W.M. Lonsdale, H. Evans, M. Clout, M. and F.A. Bazzaz. 2000. Biotic invasions: Causes, epidemiology, global consequences, and control. Ecological Applications 10(3):689–710. Maher, C.T., C.R. Nelson, A.J. Larson, and A. Sala. 2018. Ecological effects and effectiveness of silvicultural resto- ration treatments in whitebark pine forests. Forest Ecology and Management 429:534–548. Maloy, O.C. 1997. White pine blister rust control in North America: A case history. Annual Review of Phytopathology 35:87–109. Margoles, D.S. 2011. Mountain Pine Beetle–Whitebark Pine Dynamics in a Subalpine Ecosystem of the Pioneer Mountains, Southwest Montana. Master (M.S.) Thesis, University of Minnesota. Martı́n-Trillo, M., and J.M. Martı́nez-Zapater. 2002. Growing up fast: Manipulating the generation time of trees. Current Opinion in Biotechnology 13(2):151–155. Maxwell, D.L., E.L. Kruger, and G.R. Stanosz. 1997. Effects of water stress on colonization of poplar stems and excised leaf disks by Septoria musiva. Phytopathology 87(4):381–388. McCullough, D.G., and R.J. Mercader. 2012. Evaluation of potential strategies to SLow Ash Mortality (SLAM) caused by emerald ash borer (Agrilus planipennis): SLAM in an urban forest. International Journal of Pest Man- agement 58(1):9–23. McCullough, D.G., R.J. Mercader, and N.W. Siegert. 2015. Developing and integrating tactics to slow ash (Oleaceae) mortality caused by emerald ash borer (Coleoptera: Buprestidae). The Canadian Entomologist 147(3):349–358. McDonald, B.A., and C. Linde. 2002. Pathogen population genetics, evolutionary potential and durable resistance. Annual Review of Phytopathology 40:349–379 McDonald, G., P. Zambino, and R. Sniezko. 2004. Breeding rust-resistant five-needle pines in the western United States. Pp. 28–50 in Breeding and Genetic Resources of Five-needle Pines: Growth, Adaptability, and Pest Resistance, R.A. Sniezko, S. Samman, S.E. Schlarbaum, and H.B. Kriebel, eds. Fort Collins, CO: U.S. Forest Service. McKinney, S.T., and D.F. Tomback. 2007. The influence of white pine blister rust on seed dispersal in whitebark pine. Canadian Journal of Forest Research 37(6):1044–1057. Mercader, R.J., D.G. McCullough, A.J. Storer, J.M. Bedford, R. Heyd, N.W. Siegert, S. Katovich, and T.M. Poland. 2016. Estimating local spread of recently established emerald ash borer, Agrilus planipennis, infestations and the potential to influence it with a systemic insecticide and girdled ash trees. Forest Ecology and Management 366:87–97. Mercader, R.J., N.W. Siegert, A.M. Liebhold, and D.G. McCullough. 2011. Simulating the effectiveness of three potential management options to slow the spread of emerald ash borer (Agrilus planipennis) populations in localized outlier sites. Canadian Journal of Forest Research 41(2):254–264. Merkle, S.A., and J.F.D. Dean. 2000. Forest tree biotechnology. Current Opinion in Biotechnology 11(3):298–302. 82 Prepublication Copy

Mitigating Threats to Forest Health Merkle, S.A., A.R. Tull, H.J. Gladfelter, P.M. Montello, J.E. Mitchell, C. Ahn, and R.D. McNeill. 2017. Somatic embryogenesis and cryostorage for conservation and restoration of threatened forest trees. Pp. 113–116 in Proceedings of Workshop on Gene Conservation of Tree Species–Banking on the Future, R.A. Sniezko, G. Man, V. Hipkins, K. Woeste, D. Gwaze, J.T.Kliejunas, and B.A. McTeague, tech. coords. Portland, OR: USDA–FS. Merkle, S.A., A.T. Wiecko, and A.B. Watson-Pauley. 1991. Somatic embryogenesis in American chestnut. Canadian Journal of Forest Research 21(11):1698–1701. Milgroom, M.G., and P. Cortesi. 2004. Biological control of chestnut blight with hypovirulence: A critical analysis. Annual Review of Phytopathology 42:311–338. Mundt, C.C., 2014. Durable resistance: A key to sustainable management of pathogens and pests. Infection, Genetics and Evolution 27:446–455. NASEM (National Academies of Sciences, Engineering, and Medicine). 2016. Gene Drives on the Horizon: Advanc- ing Science, Navigating Uncertainty, and Aligning Research with Public Values. Washington, DC: The National Academies Press. Neuhaus, G., and G. Spangenberg. 1990. Plant transformation by microinjection techniques. Physiologia Plantarum 79(1):213–217. Newcombe, G., and M. Ostry. 2001. Recessive resistance to Septoria stem canker of hybrid poplar. Phytopathology 91(11):1081–1084. Newhouse, A. 2018. Transgenic American Chestnuts for Potential Forest Restoration: Scientific Successes, Regula- tory Challenges. Presentation at the Genetic Engineering and Society Center Colloquium, April 24, North Car- olina State University, Raleigh, NC. Available at https://research.ncsu.edu/ges/colloquium/2017-18-colloqu ia/. Accessed November 27, 2018. Newhouse, A.E., L.D. McGuigan, K.A. Baier, K.E. Valletta, W.H. Rottmann, T.J. Tschaplinski, C.A. Maynard, and W.A. Powell. 2014a. Transgenic American chestnuts show enhanced blight resistance and transmit the trait to T1 progeny. Plant Science 228:88–97. Newhouse, A.E., J.E. Spitzer, C.A. Maynard, and W.A. Powell. 2014b. Chestnut leaf inoculation assay as a rapid predictor of blight susceptibility. Plant Disease 98(1):4–9. Nguyen, V.P., J.-S. Cho, J.-H. Lee, M.-H. Kim, Y.-I. Choi, E.-J. Park, W.-C. Kim, S. Hwang, K.-H. Han, and J.-H. Ko. 2017. Identification and functional analysis of a promoter sequence for phloem tissue specific gene expres- sion from Populus trichocarpa. Journal of Plant Biology 60(2):129–136. Ostry, M.E. 1987. Biology of Septoria musiva and Marssonina brunnea in hybrid Populus plantations and control of Septoria canker in nurseries. European Journal of Forest Pathology 17(3):158–165. Ostry, M.E., G. Laflamme, and S.A. Katovich. 2010. Silvicultural approaches for management of eastern white pine to minimize impacts of damaging agents. Forest Pathology 40(3–4): 332–346. Ostry, M.E., and K.T. Ward. 2003. Field performance of Populus expressing somaclonal variation in resistance to Septoria musiva. Plant Science 164(1):1–8. Padovan, A., A. Keszei, W.J. Foley and C. Külheim. 2013. Differences in gene expression with a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory. BMC Plant Biology 13:29. Palla, K.J., and P.M. Pijut. 2015. Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotyls. Plant Cell, Tissue and Organ Culture 120(2):631–641. Palloix, A., V. Ayme, and B. Moury. 2009. Durability of plant major resistance genes to pathogens depends on the genetic background, experimental evidence and consequences for breeding strategies. New Phytologist 183(1): 190–199. Parlevliet, J.E., and J.C. Zadoks. 1977. The integrated concept of disease resistance: A new view including horizontal and vertical resistance in plants. Euphytica 26(1):5–21. Perkins, D.L., C.L. Jorgensen, and M.J. Rinella. 2015. Verbenone decreases whitebark pine mortality throughout a mountain pine beetle outbreak. Forest Science 61(4):747–752. Pickett, C.H., J.C. Ball, K.C. Casanave, K.M. Klonsky, K.M. Jetter, L.G. Bezark, and S.E. Schoenig. 1996. Establish- ment of the ash whitefly parasitoid Encarsia inaron (Walker) and its economic benefit to ornamental street trees in California. Biological Control 6(2):260–272. Plomion, C., J.-M. Aury, J. Amselem, T. Leroy, F. Murat, S. Duplessis, S. Faye, N. Francillonne, K. Labadie, G. Le Provost, I. Lesur, J. Bartholomé, P. Faivre-Rampant, A. Kohler, J.-C. Leplé, N. Chantret, J. Chen, A. Diévart, T. Alaeitabar, V. Barbe, C. Belser, H. Bergès, C. Bodénès, M.-B. Bogeat-Triboulot, M.-L. Bouffaud, B. Brachi, E. Chancerel, D. Cohen, A. Couloux, C. Da Silva, C. Dossat, F. Ehrenmann, C. Gaspin, J. Grima-Pettenati, E. Guichoux, A. Hecker, S. Herrmann, P. Hugueney, I. Hummel, C. Klopp, C. Lalanne, M. Lascoux, E. Lasserre, A. Lemainque, M.-L. Desprez-Loustau, I. Luyten, M.-A. Madoui, S. Mangenot, C. Marchal, F. Maumus, J. Prepublication Copy 83

Forest Health and Biotechnology: Possibilities and Considerations Mercier, C. Michotey, O. Panaud, N. Picault, N. Rouhier, O. Rué, C. Rustenholz, F. Salin, M. Soler, M. Tarkka, A. Velt, A.E. Zanne, F. Martin, P. Wincker, H. Quesneville, A. Kremer, and J. Salse. 2018. Oak genome reveals facets of long lifespan. Nature Plants 4 (7): 440–452. Poland, T.M., and D.G. McCullough. 2006. Emerald ash borer: Invasion of the urban forest and the threat to North America’s ash resource. Journal of Forestry 104(3):118–124. Polin, L.D., H. Liang, R.E. Rothrock, M. Nishii, D.L. Diehl, A.E. Newhouse, C.J. Nairn, W.A. Powell, and C.A. Maynard. 2006. Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos. Plant Cell, Tissue and Organ Culture 84(1):69–79. Powell W.A. 2014. The American chestnut’s genetic rebirth. Scientific American 310:68–73. Prasad, A.M., L.R. Iverson, M.P. Peters, J.M. Bossenbroek, S.N. Matthews, S.D. Syndor, and M.W. Schwartz. 2010. Modeling the invasive emerald ash borer risk of spread using a spatially explicit cellular model. Landscape Ecology 25(3):353–369. Qin, R., and J.M. LeBoldus. 2014. The infection biology of Sphaerulina musiva: Clues to understanding a forest pathogen. PLoS ONE 9(7):e103477. Raffa, K.F., B.H. Aukema, B.J. Bentz, A.L. Carroll, J.A. Hicke, M.G. Turner, and W.H. Romme. 2008. Cross-scale drivers of natural disturbance prone to anthropogenic amplification: The dynamics of bark beetle eruptions. Bioscience 58(6):501–517. Raffa, K.F., E.N. Powell, and P.A. Townsend. 2013. Temperature-driven range expansion of an irruptive insect height- ened by weakly coevolved plant defenses. Proceedings of the National Academy of Sciences of the United States of America 110(6):2193–2198. Rasche, L., L. Fahse, and H. Bugmann. 2013. Key factors affecting the future provision of tree-based forest ecosystem goods and services. Climatic change 118(3–4):579–593. Remington, D.L., and D.M. O’Malley. 2000. Whole-genome characterization of embryonic stage inbreeding depres- sion in a selfed loblolly pine family. Genetics 155(1):337–348. Resende, M.F.R., P. Muñoz, J.J. Acosta, G.F. Peter, J.M. Davis, D. Grattapaglia, M.D.V. Resende, and M. Kirst. 2012. Accelerating the domestication of trees using genomic selection: Accuracy of prediction models across ages and environments. New Phytologist 193(3):617–624. Rigsby, C.M., D.A. Herms, P. Bonello, and D. Cipollini. 2016. Higher activities of defense-associated enzymes may contribute to greater resistance of Manchurian ash to emerald ash borer than a closely related and susceptible congener. Journal of Chemical Ecology 42(8):782–792. Riyal, D. 2011. Development of Mutation Based Breeding Technology in Forest Tree Species. M.S. thesis, Simon Fraser University. Rose, R. 2018. USDA, Animal and Plant Health Inspection Service, Plant Protection and Quarantine: Biological con- trol permitting overview. Webinar presentation to the National Academies of Sciences, Engineering, and Med- icine Committee on the Potential for Biotechnology to Address Forest Health, February 23. Roy, B.A., H.M. Alexander, J. Davidson, F.T. Campbell, J.J. Burdon, R. Sniezko, and C. Brasier. 2014. Increasing forest loss worldwide from invasive pests requires new trade regulations. Frontiers in Ecology and the Environ- ment 12(8):457–465. Sanford, J.C., T.M. Klein, E.D. Wolf, and N. Allen. 1987. Delivery of substances into cells and tissues using a particle bombardment process. Particulate Science and Technology 5(1):27–37. Santos, C., H. Machado, I. Correia, F. Gomes, J. Gomes‐Laranjo, and R. Costa. 2015. Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance. Plant Pathology 64(4):901–910. Schoenweiss, K., and A. Meier-Dinkel. 2005. In vitro propagation of selected mature trees and juvenile embryo- derived cultures of common ash (Fraxinus excelsior L.). Propagation of Ornamental Plants 5(3):137–145. Schwandt, J.W., I.B. Lockman, J.T. Kliejunas, and J.A. Muir. 2010. Current health issues and management strategies for white pines in the western United States and Canada. Forest Pathology 40(3–4):226–250. Sharov, A.A., D. Leonard, A.M. Liebhold, E.A. Roberts, and W. Dickerson. 2002. “Slow the spread”: A national program to contain the gypsy moth. Journal of Forestry 100(5):30–36. Shelton, A.M., J. Zhao, and R.T. Roush. 2002. Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annual Reviews in Entomology 47:845–881. Showalter, D.N., K.F. Raffa, R.A. Sniezko, D.A. Herms, A.M. Liebhold, J.A. Smith, and P. Bonello. 2018. Strategic development of tree resistance against forest pathogen and insect invasions in defense-free space. Frontiers in Ecology and Evolution 6:124. Simmonds, N. 1991. Genetics of horizontal resistance to diseases of crops. Biological Reviews 66(2):189–241. Sisco, P.H. 2004. Breeding blight resistant American chestnut trees. Journal of The American Chestnut Foundation 18:15. 84 Prepublication Copy

Mitigating Threats to Forest Health Six, D.L., C. Vergobbi, and M. Cutter. 2018. Are survivors different? Genetic-based selection of trees by mountain pine beetle during a climate change-driven outbreak in a high-elevation pine forest. Frontiers in Plant Science 9:993. Sniezko, R.A., and J. Koch. 2017. Breeding trees resistant to insects and diseases: Putting theory into application. Biological Invasions 19(11):3377–3400. Sniezko, R.A., A. Bower, and J. Danielson. 2000. A comparison of early field results of white pine blister rust re- sistance in sugar pine and western white pine. HortTechnology 10(3):519–522. Sniezko, R.A., A. Kegley, R. Danchok, and S. Long. 2007. Variation in resistance to white pine blister rust among whitebark pine families from Oregon and Washington—early results and implications for conservation. Pp. 82– 97 in Proceedings of the Conference Whitebark Pine: A Pacific Coast Perspective, E.M. Goheen and R.A. Sniezko, tech. cords. Portland, OR: U.S. Forest Service. Sniezko, R.A., A. Kegley, R. Danchok, and S. Long. 2018. Blister rust resistance in whitebark pine (Pinus albicau- lis)—early results following artificial inoculation of seedlings from Oregon, Washington, Idaho, Montana, Cal- ifornia, and British Columbia seed sources. Pp. 129–135 in Proceedings of the IUFRO Joint Conference: Ge- netics of Five-needle Pines, Rusts of Forest Trees, and Strobusphere, A.W. Schoettle, R.A. Sniezko, and J.T. Kliejunas, eds. Fort Collins, CO: U.S. Forest Service. Sniezko, R.A., J. Hamlin, and E.M. Hansen. 2012. Operational program to develop Phytophthora lateralis-resistant populations of Port-Orford-cedar (Chamaecyparis lawsoniana). Pp. 65–79 in Proceedings of the Fourth Inter- national Workshop on the Genetics of Host–Parasite Interactions in Forestry: Disease and Insect Resistance in Forest Trees, R.A. Sniezko, A.D. Yanchuk, J.T. Kliejunas, K.M. Palmieri, J.M. Alexander, and S.J. Frankel, tech. cords. Albany, CA: U.S. Forest Service. Sniezko, R.A., M.F. Mahalovich, A.W. Schoettle, and D.R. Vogler. 2011. Past and current investigations of the genetic resistance to Cronartium ribicola in high-elevation five-needle pines. Pp. 246–264 in The Future of High-ele- vation, Five-needle White Pines in Western North America: Proceedings of the High Five Symposium, R.E. Keane, D.F. Tomback, M.P. Murray, and C.M. Smith, eds. Fort Collins, CO: U.S. Forest Service. Sniezko, R.A., J. Smith, J.-J. Liu, and R.C. Hamelin. 2014. Genetic resistance to fusiform rust in southern pines and white pine blister rust in white pines—A contrasting tale of two rust pathosystems—Current status and future prospects. Forests 5(9):2050–2083. Sollars, E.S.A., A.L. Harper, L.J. Kelly, C.M. Sambles, R.H. Ramirez-Gonzalez, D. Swarbreck, G. Kaithakottil, E.D. Cooper, C. Uauy, L. Havlickova, G. Worswick, D.J. Studholme, J. Zohren, D. L. Salmon, B.J. Clavijo, Y. Li, Z. He A. Fellgett, L.V. McKinney, L.R. Nielsen, G.C. Douglas, E.D. Kjær, J.A. Downie, D. Boshier, S. Lee, J. Clark, M. Grant, I. Bancroft, M. Caccamo, R.J.A. Buggs. 2017. Genome sequence and genetic diversity of European ash trees. Nature 541(7636):212–216. Springer, P.S. 2000. Gene traps: Tools for plant development and genomics. Plant Cell 12(7):1007–1020. Steiner, K.C., J.W. Westbrook, F.V. Hebard, L.L. Georgi, W.A. Powell, and S.F. Fitzsimmons S.F. 2017. Rescue of American chestnut with extraspecific genes following its destruction by a naturalized pathogen. New Forests 48(2):317–336. Stevens, M.E., and P.M. Pijut. 2014. Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda. Plant Cell Reports 33(6):861–870. Stevens, K.A., J.L. Wegrzyn, A. Zimin, D. Puiu, M. Crepeau, C. Cardeno, R. Paul, D. Gonzalez-Ibeas, M. Koriabine, A.E. Holtz-Morris, P.J. Martínez-García, U.U. Sezen, G. Marçais, K. Jermstad, P.E. McGuire, C.A. Loopstra, J.M. Davis, A. Eckert, P. de Jong, J.A. Yorke, S.L. Salzberg, D.B. Neale, and C.H. Langley. 2016. Sequence of the sugar pine megagenome. Genetics 204(4):1613–1626. Stoddard, E.M., and A.E. Moss. 1913. The Chestnut Bark Disease, Bulletin 178. New Haven: The Connecticut Agri- cultural Experiment Station. Suprasanna, P., T.R. Ganapathi, and V.A. Bapat. 2005. Genetic transformation of woody plants using embryogenic cultures. Journal of New Seeds 7(2):17–35. Tang, G., B.J. Reinhart, D.P. Bartel, and P.D. Zamore. 2003. A biochemical framework for RNA silencing in plants. Genes & Development 17(1):49–63. Telford, A., S. Cavers, R.A. Ennos, and J.E. Cottrell. 2015. Can we protect forests by harnessing variation in resistance to pests and pathogens? Forestry 88(1):3–12. Thynne, E., I.M.L. Saur, J. Simbaqueba, H.A. Ogilvie, Y. Gonzalez-Cendales, O. Mead, A. Taranto, A.-M. Catanza- riti, M.C. McDonald, B. Schwessinger, D.A. Jones, J.P. Rathjen, and P.S. Solomon. 2017. Fungal phytopatho- gens encode functional homologues of plant rapid alkalinization factor (RALF) peptides. Molecular Plant Pa- thology 18(6):811–824. Prepublication Copy 85

Forest Health and Biotechnology: Possibilities and Considerations Tobin, P.C., J.M. Kean, D.M. Suckling, D.G. McCullough, D.A. Herms, and L.D. Stringer. 2014. Determinants of successful arthropod eradication programs. Biological Invasions 16(2):401–414. Tomback, D.F., A.J. Anderies, K.S. Carsey, M.I. Powell, and S. Mellman-Brown. 2001. Delayed seed gemination in whitebark pine and regeneration pattern following the Yellowstone fires. Ecology 82(9): 2587–2600. Tsai, C.J., and L.J. Xue. 2015. CRISPRing into the woods. GM Crops & Food 6(4):206–215. Tscharntke, T., R. Bommarco, Y. Clough, T.O. Crist, D. Kleijn, T.A. Rand, J.M. Tylianakis, J.M., S. van Nouhuys, and S. Vidal. 2008. Reprint of “Conservation biological control and enemy diversity on a landscape scale” [Biol. Control 43 (2007) 294–309]. Biological Control 45(2):238–253. Tuskan, G.A., S. DiFazio, S. Jansson, J. Bohlmann, I. Grigoriev, U. Hellsten, N. Putnam, S. Ralph, S. Rombauts, A. Salamov, J. Schein, L. Sterck, A. Aerts, R.R. Bhalerao, R.P. Bhalerao, D. Blaudez, W. Boerjan, A. Brun, A. Brunner, V. Busov, M. Campbell, J. Carlson, M. Chalot, J. Chapman, G.-L. Chen, D. Cooper, P.M. Coutinho, J. Couturier, S. Covert, Q. Cronk, R. Cunningham, J. Davis, S. Degroeve, A. Déjardin, C. dePamphilis, J. Detter, B. Dirks, I. Dubchak, S. Duiplessis, J. Ehlting, B. Ellis, K. Gendler, D. Goodstein, M. Gribskov, J. Grimwood, A. Groover, L. Gunter, B. Hamberger, B. Heinze, Y. Helariutta, B. Henrissat, D. Holligan, R. Holt, W. Huang, N. Islam-Faridi, S. Jones, M. Jones-Rhoades, R. Jorgensen, C. Joshi, J. Kangasjärvi, J. Karlosson, C. Kelleher, R. Kirkpatrick, M. Kirst, A. Kohler, U. Kalluri, F. Larimer, J. Leebens-Mack, J.-C. Leplé, P. Locascio, Y. Lou, S. Lucas, F. Martin, B. Montanini, C. Napoli, D.R. Nelson, C. Nelson, K. Nieminen, O. Nilsson, V. Pereda, G. Peter, R. Philippe, G. Pilate, A. Poliakov, J. Razumovskaya, P. Richardson, C. Rinaldi, K. Ritland, P. Rouzé, D. Ryaboy, J. Schmutz, J. Schrader, B. Segerman, H. Shin, A. Siddiqui, F. Sterkyk, A. Terry, C.-J. Tsai, E. Uber- bacher, P. Unneberg, J. Vahala, K. Wall, S. Wessler, G. Yang, T. Yin, C. Douglas, M. Marra, G. Sandberg, Y. Van de Peer, and D. Rokhsar. 2006. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313(5793):1596–1604. Tuzun, S. 2001. The relationship between pathogen-induced systemic resistance (ISR) and multigenic (horizontal) resistance in plants. European Journal of Plant Pathology 107(1):85–93. USDA-APHIS (U.S. Department of Agriculture Animal and Plant Health Inspection Service). 2007. Availability of an Environmental Assessment for the Proposed Release of Three Parasitoids for the Biological Control of the Emerald Ash Borer Agrilus planipennis in the Continental United States. Federal Register 72:28947–28948. Venette, R.C., D.J. Kriticos, R.D. Magarey, F.H. Koch, R.H. Baker, S.P. Worner, N.N. Gómez Raboteaux, D.W. McKenney, E.J. Dobesberger, D. Yemshanov, and P.J. De Barro. 2010. Pest risk maps for invasive alien species: a roadmap for improvement. BioScience 60(5):349–362. Wainhouse, D. 2005. Ecological Methods in Forest Pest Management. Oxford: Oxford University Press. Wang, H., M. La Russa, and L.S. Qi. 2016. CRISPR/cas9 in genome editing and beyond. Annual Review of Biochem- istry 85(1):227–264. Welch, A.J., A.J. Stipanovic, C.A. Maynard, and W.A. Powell. 2007. The effects of oxalic acid on transgenic Castanea dentata callus tissue expressing oxalate oxidase. Plant Science 172(3):488–496. Westbrook, J. 2017. Restoration of American chestnut: A marriage of breeding and biotechnology. Webinar presen- tation to the National Academies of Sciences, Engineering, and Medicine Committee on the Potential for Bio- technology to Address Forest Health, December 12. Westbrook, J.W., J.B. James, S. Lucas, F.V. Hebard, J. Frampton, and S.N. Jeffers. 2018. Resistance to Phytophthora cinnamomi in American chestnut (Castanea dentata) backcross populations that descended from two Chinese chestnut (Castanea mollissima) sources of resistance. Plant Disease forthcoming. White, T.L., W.T. Adams, and D.B. Neale. 2007. Forest Genetics. Cambridge, MA: CABI. Whitehill, J.G.A., S.O. Opiyo, J.L. Koch, D.A. Herms, D.F. Cipollini, and P. Bonello. 2012. Interspecific comparison of constitutive ash phloem phenolic chemistry reveals compounds unique to Manchurian ash, a species resistant to emerald ash borer. Journal of Chemical Ecology 38(5):499–511. Williams, C., R.D. Barnes, and I. Nyoka. 1999. Embryonic genetic load for a neotropical conifer, Pinus patula Schiede et Deppe. Journal of Heredity 90(3):394–398. Woodbury, P.B., J.A. Laurence, and G.W. Hudler. 1994. Chronic ozone exposure increases the susceptibility of hybrid Populus to disease caused by Septoria musiva. Environmental Pollution 86(1):109–114. Woodcock, P, J.E. Cottrell, R.J.A. Buggs, and C.P. Quine. 2017. Mitigating pest and pathogen impacts using resistant trees: A framework and overview to inform development and deployment in Europe and North America. For- estry 91(1):1–16. Wu, S.H., S.X. Zhang, J.Q. Chao, X.M. Deng, Y.Y. Chen, M.J. Shi, and W.M. Tian. 2016. Transcriptome analysis of the signalling networks in coronatine-induced secondary laticifer differentiation from vascular cambia in rubber trees. Scientific Reports 6:36384. 86 Prepublication Copy

Mitigating Threats to Forest Health Yang, D., L. Bernier, and M. Dessureault. 1994. Biological control of Septoria leaf spot of poplar by Phaeotheca dimorphospora. Plant Disease 78(8):821–825. Zayed, M.Z., W.-S. Ho, S.-L. Pang, and F.B. Ahmad 2014. EMS-induced mutagenesis and DNA polymorphism assessment through ISSR markers in Neolamarckia cadamba (kelampayan) and Leucaena leucocephala (petai belalang). European Journal of Experimental Biology 4(4):156–163. Zhang, B., A. Newhouse, L. McGuigan, C. Maynard, and W. Powell. 2011. Agrobacterium-mediated co-transfor- mation of American chestnut (Castanea dentata) somatic embryos with a wheat oxalate oxidase gene. BMC Proceedings 5(Suppl 7):O43. Zhang, B., A.D. Oakes, A.E. Newhouse, K.M. Baier, C.A. Maynard and W.A. Powell. 2013. A threshold level of oxalate oxidase transgene expression reduces Cryphonectria parasitica-induced necrosis in a transgenic Amer- ican chestnut (Castanea dentata) leaf bioassay. Transgenic Research 22(5):973–982. Zhang, D.-X., M.J. Spiering, A.L. Dawe, and D.L. Nuss. 2014. Vegetative incompatibility loci with dedicated roles in allorecognition restrict mycovirus transmission in chestnut blight fungus. Genetics 197(2):701–714. Zhang, D.-X., and D.L. Nuss. 2016. Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic gene. Proceedings of the National Academy of Sciences of the United States of America 113:2062–2067. Zhang, X.Y., Q. Lu, R.A. Sniezko, R.Q. Song, and G. Man. 2010. Blister rusts in China: Hosts, pathogens, and man- agement. Forest Pathology 40(3–4):369–381. Zimin, A., K.A. Stevens, M.W. Crepeau, A. Holtz-Morris, M. Koriabine, G. Marçais, D. Puiu, M. Roberts, J.L. Wegrzyn, P.J. de Jong, D.B. Neale, S.L. Salzberg, J.A. Yorke, and C.H. Langley. 2014. Sequencing and assem- bly of the 22-gb loblolly pine genome. Genetics 196(3):875–890. Zupan, J.R., and P. Zambryski. 1995. Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiology 107(4):1041–1047. Prepublication Copy 87

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The American chestnut, whitebark pine, and several species of ash in the eastern United States are just a few of the North American tree species that have been functionally lost or are in jeopardy of being lost due to outbreaks of pathogens and insect pests. New pressures in this century are putting even more trees at risk. Expanded human mobility and global trade are providing pathways for the introduction of nonnative pests for which native tree species may lack resistance. At the same time, climate change is extending the geographic range of both native and nonnative pest species.

Biotechnology has the potential to help mitigate threats to North American forests from insects and pathogens through the introduction of pest-resistant traits to forest trees. However, challenges remain: the genetic mechanisms that underlie trees’ resistance to pests are poorly understood; the complexity of tree genomes makes incorporating genetic changes a slow and difficult task; and there is a lack of information on the effects of releasing new genotypes into the environment.

Forest Health and Biotechnology examines the potential use of biotechnology for mitigating threats to forest tree health and identifies the ecological, economic, and social implications of deploying biotechnology in forests. This report also develops a research agenda to address knowledge gaps about the application of the technology.

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