Verbenone Plus Reduces Levels of Tree Mortality Attributed to Mountain Pine Beetle Infestations in Whitebark Pine, a Tree Species of Concern

In western North America, recent outbreaks of the mountain pine beetle, Dendroctonus ponderosae Hopkins, have been severe, long-lasting and well-documented. We review previous research that led to the identification of Verbenone Plus, a novel four-component semiochemical blend [acetophenone, (E)-2-hexen-1-ol + ( )-2-hexen-1ol, and (–)-verbenone] that has been demonstrated to inhibit the response of a closely-related bark beetle species, western pine beetle, D. brevicomis LeConte, to attractant-baited traps and trees. In this study, we evaluate the efficacy of Verbenone Plus for protecting stands of whitebark pine, Pinus albicaulis Engelm., a species of concern being considered for listing as a threatened and endangered species, from mortality attributed to D. ponderosae infestations in the central Sierra Nevada, California, USA. The experimental design was completely randomized with two treatments (untreated control, Verbenone Plus) and four replicates (0.4-ha square plots) per treatment. A total of 450 trees were killed by D. ponderosae, 377 were P. albicaulis and 73 were lodgepole pine, P. contorta Dougl. ex Laws. Significantly fewer pines (P. albicaulis and P. contorta) and P. albicaulis (only) were killed by D. ponderosae on Verbenone Plus-treated plots compared to the untreated control. On average, there was ~78% reduction in tree mortality attributed to Verbenone Plus. We discuss the implications of these and other results to the development of Verbenone Plus as a semiochemical-based tool for tree protection. to the list of candidate species eligible for protection, and will continue to review its status on an annual basis [8]. Pinus albicaulis plays a major ecological role in the functioning of high elevation ecosystems, surviving conditions that are often too cold, too dry and too windy for many other tree species [5]. Pinus albicaulis is considered a keystone species in the subalpine environment, stabilizing soils, moderating and regulating runoff, and facilitating the establishment and survival of other species [5,9]. Due to the slow growth and maturation of P. albicaulis, and the unique ecological services this species provides, protection of P. albicaulis from D. ponderosae is desirable, but challenging. Development of environmentally-friendly (e.g., biopesticides) and portable methods of tree protection are needed given the remote and sensitive nature of the subalpine environments where P. albicaulis persists. Verbenone (4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-one) is an anti aggregation pheromone of D. ponderosae, western pine beetle, D. brevicomis LeConte, and southern pine beetle, D. frontalis Zimmerman [10], and is produced by auto-oxidation of the host monoterpene α-pinene via the intermediary compounds cisand trans-verbenol [11], by the beetles themselves [12], and/or through degradation of host material typically by microorganisms associated with bark beetles [13-15]. Because of its behavioral activity, as demonstrated in Journal of Biofertilizers & Biopesticides J o u r n a l o f B iof ertilizers & Bipesti c i d e s


Abstract
In western North America, recent outbreaks of the mountain pine beetle, Dendroctonus ponderosae Hopkins, have been severe, long-lasting and well-documented. We review previous research that led to the identification of Verbenone Plus, a novel four-component semiochemical blend [acetophenone, (E)-2-hexen-1-ol + ( )-2-hexen-1ol, and (-)-verbenone] that has been demonstrated to inhibit the response of a closely-related bark beetle species, western pine beetle, D. brevicomis LeConte, to attractant-baited traps and trees. In this study, we evaluate the efficacy of Verbenone Plus for protecting stands of whitebark pine, Pinus albicaulis Engelm., a species of concern being considered for listing as a threatened and endangered species, from mortality attributed to D. ponderosae infestations in the central Sierra Nevada, California, USA. The experimental design was completely randomized with two treatments (untreated control, Verbenone Plus) and four replicates (0.4-ha square plots) per treatment. A total of 450 trees were killed by D. ponderosae, 377 were P. albicaulis and 73 were lodgepole pine, P. contorta Dougl. ex Laws. Significantly fewer pines (P. albicaulis and P. contorta) and P. albicaulis (only) were killed by D. ponderosae on Verbenone Plus-treated plots compared to the untreated control. On average, there was ~78% reduction in tree mortality attributed to Verbenone Plus. We discuss the implications of these and other results to the development of Verbenone Plus as a semiochemical-based tool for tree protection.
to the list of candidate species eligible for protection, and will continue to review its status on an annual basis [8].
Pinus albicaulis plays a major ecological role in the functioning of high elevation ecosystems, surviving conditions that are often too cold, too dry and too windy for many other tree species [5]. Pinus albicaulis is considered a keystone species in the subalpine environment, stabilizing soils, moderating and regulating runoff, and facilitating the establishment and survival of other species [5,9]. Due to the slow growth and maturation of P. albicaulis, and the unique ecological services this species provides, protection of P. albicaulis from D. ponderosae is desirable, but challenging. Development of environmentally-friendly (e.g., biopesticides) and portable methods of tree protection are needed given the remote and sensitive nature of the subalpine environments where P. albicaulis persists.
Verbenone (4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-one) is an anti aggregation pheromone of D. ponderosae, western pine beetle, D. brevicomis LeConte, and southern pine beetle, D. frontalis Zimmerman [10], and is produced by auto-oxidation of the host monoterpene α-pinene via the intermediary compounds cis-and trans-verbenol [11], by the beetles themselves [12], and/or through degradation of host material typically by microorganisms associated with bark beetles [13][14][15]. Because of its behavioral activity, as demonstrated in numerous trapping bioassays, verbenone has been evaluated as a tool for mitigating coniferous tree mortality due to bark beetle infestations in western North America. Efforts have concentrated on individual tree [e.g., [16][17][18] or small-scale (e.g., <4 ha) stand protection, primarily from D. ponderosae [e.g., [19][20][21][22]. Results have been favorable, but inconsistent. Negative results have been linked to photoisomerization of verbenone to behaviorally inactive chrysanthenone [23]; inconsistent or inadequate release [24]; rapid dispersal of verbenone [25]; and/or limitations in the range of inhibition of verbenone [26], particularly when D. ponderosae populations were high [19][20][21]. A lack of efficacy may also be due to the complexity of the host selection process, which involves other visual and olfactory cues produced by hosts, non hosts and competing species [27]. Verbenone was first registered by the U.S. Environmental Protection Agency (licensed for sale and distribution) in December 1999 to control D. frontalis in southern forests. Since then, the label has been expanded to include D. ponderosae and D. brevicomis in forests, recreational and municipal settings, and in rights of way and other easements. Verbenone is generally deployed in individual passive release devices (pouches) by hand application to the tree bole or in bead, flake and sprayable formulations by ground or aerial application [10].
Verbenone has been found ineffective for protecting individual P. ponderosa [18,28] and P. ponderosa stands [29] from D. brevicomis infestations. As a result, based on the semiochemical-diversity hypothesis [30], Shepherd et al. [27] suggested that synthetic verbenone should be deployed with other beetle-derived or non host cues that more accurately reflect the complexity of the olfactory environment in forests. In the context of pest management, a diverse array of chemical cues and signals may disrupt bark beetle searching more than high doses of a single semiochemical (e.g., verbenone) or even mixtures of semiochemicals intended to mimic one type of signal (e.g., antiaggregation pheromones), because they represent heterogeneous stand conditions to foraging insects [27,30]. Fettig et al. [31] reported that combinations of bark volatiles [benzaldehyde, benzyl alcohol, (E)conophthorin, guaiacol, nonanal, and salicylaldehyde], three green leaf volatiles [(E)-2-hexenal, (E)-2-hexen-1-ol, and (E)-2-hexen-1-ol], or the nine compounds combined did not affect the response of D. brevicomis to attractant-baited traps. However, when the bark and green leaf volatiles were combined with (-)-verbenone, they reduced trap catches to levels significantly below that of verbenone alone. A ninecomponent blend [benzyl alcohol, benzaldehyde, guaiacol, nonanal, salicylaldehyde, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol and (-)-verbenone] reduced trap catch by 87% compared to the attractantbaited control [31]. Based on this work, Fettig et al. [32] were first to demonstrate the successful application of a semiochemical-based tool for protecting P. ponderosa from mortality attributed to D. brevicomis. Additional research confirmed the effect [33], but initial blends were complex and likely not feasible for operational use. in several studies [28,34]. The objective of this study was to determine the effectiveness of Verbenone Plus for protecting P. albicaulis from mortality attributed to D. ponderosae in California, USA.  (Table  1). Plots were located in stands with a mean stand density of 48.7 m²/ha of which ~65% was P. albicaulis with the remainder P. contorta (Table  2).

Materials and Methods
Semiochemicals were hand-applied in a ~10.6 by 10.6 m grid (50 U/plot) to the nearest tree at ~2 m in height on 10 June 2010 and remained throughout the seasonal flight period of D. ponderosae [35]. Treatments were removed and plots assessed for D. ponderosae   Analyses were limited to trees successfully mass attacked by D. ponderosae during the treatment period. A tree was considered successfully mass attacked, and therefore killed by D. ponderosae if boring dust surrounded the root collar, and/or the phloem and sapwood were discolored, the bark separated readily from the sapwood, and adult (parent) galleries and larval mines were visible following bark removal. Tests of normality and equal variance were conducted to confirm data met assumptions of normality and homoscedasticity prior to analysis (SigmaStat Version 12.0, Systat Software Inc., San Jose, California, USA). The mean percentages of trees (P. albicaulis and P. contorta) and of P. albicaulis (only) killed by D. ponderosae were compared by t-test using α=0.05 (SigmaStat Version 12.0).

Results
Among all plots, a total of 469 trees were attacked by D. ponderosae. However, 19 trees exhibited strip attacks (i.e., a partial attack of the tree bole typically insufficient to cause tree mortality), and therefore were excluded from our analyses. Of the 450 trees that were killed by D. ponderosae, 377 were P. albicaulis and 73 were P. contorta. At the plot level, tree mortality ranged from 0 trees (plot 5, Verbenone Plustreated) to 139 trees (plot 7, untreated control), and from 0% to 36.6% of trees, respectively. In the untreated control, levels of tree mortality exceeded 15% on all plots. For P. albicaulis, mortality ranged from 0 trees (plot 5, Verbenone Plus-treated) to 112 trees (plot 7, untreated control), and from 0% to 41.5% of P. albicaulis, respectively. A significantly lower percentage of trees (P. albicaulis and P. contorta) were killed by D. ponderosae on Verbenone Plus-treated compared to untreated control plots (t = -4.25, P = 0.005) (Figure 2). The effect was consistent for P. albicaulis as significantly fewer P. albicaulis died on Verbenone Plus-treated plots (t = -4.04, P = 0.007) (Figure 2). On average, there was ~78% reduction in tree mortality attributed to the application of Verbenone Plus in P. albicaulis stands.

Discussion
This paper is the first report on the effectiveness of Verbenone Plus for protecting P. albicaulis from mortality attributed to D. ponderosae. In 2008, we examined the effect of Verbenone Plus on the response of D. ponderosae to attractant-baited traps in P. contorta stands in Utah, USA, but the experiment failed to produce meaningful results due to adverse weather conditions that hampered D. ponderosae flight. In that experiment, 4.9 ± 1.5 and 0.2 ± 0.1 D. ponderosae (mean ± SEM, n = 42) were captured in the control and Verbenone Plus treatments, respectively. Several years of research initially resulted in the development of Verbenone Plus for protecting P. ponderosa from mortality attributed to D. brevicomis [27][28][29][31][32][33][34], where it serves as the only effective semiochemical-based tool for tree protection in that system [28].
Limited work has occurred regarding the development of semiochemical-based tools to protect P. albicaulis from D. ponderosae infestation [17,21,[36][37][38]. This is likely due to its limited commercial value [5], and that until recent years levels of tree mortality attributed to D. ponderosae in P. albicaulis forests were limited throughout much of the geographic range [9] (Figure 1). In the Sierra Nevada, P. albicaulis has experienced significant levels of tree mortality ( Figure  1), and some previous attempts to protect trees from D. ponderosae by application of verbenone have failed (e.g., June Mountain Ski Area in 2009; B. Bulaon, unpubl. data).
Warwell et al. [39] modeled the contemporary climate profiles of P. albicaulis and predicted future responses to warming. They reported rapid and large-scale declines in the area occupied by P. albicaulis. For example, the contemporary climate profile was predicted to decline by ~70% and move upward in elevation by ~330 m by 2030. By the end of this century, the contemporary climate profile of P. albicaulis was projected to decline to an area equivalent to <3% of its current distribution [39]. In 2007, the Whitebark Pine Restoration Program was initiated by the USDA Forest Service with the primary goals of protecting and enhancing P. albicaulis populations, providing adequate   regeneration, and increasing the proportion of P. albicaulis with natural resistance to white pine blister rust [40]. This should include maintenance and protection of mature, cone-bearing and diseaseresistant trees from throughout the geographic range of P. albicaulis [7]. Based on our research, we suggest additional work on Verbenone Plus should concentrate on determining optimal release rates and spacing necessary to achieve adequate levels of efficacy in other areas throughout the range of P. albicaulis (Figure 1), and comparison of the efficacy of Verbenone to Verbenone Plus within the same P. albicaulis stands. Ongoing research (not presented here) indicates that Verbenone Plus is effective for protecting P. contorta from mortality attributed to D. ponderosae (C. Fettig, unpubl. data), and its efficacy compared to verbenone alone is being evaluated in that system. Such data would be useful in facilitating commercialization of Verbenone Plus (i.e., as the only effective semiochemical-based tool for D. brevicomis) given the recent impacts of D. ponderosae to forest resources.