Microb Ecol DOI 10.1007/s00248-015-0593-8
FUNGAL MICROBIOLOGY
Effects of Temperature on Growth, Sporulation, and Competition of Mountain Pine Beetle Fungal Symbionts Melissa L. Moore & Diana L. Six
Received: 30 December 2014 / Accepted: 26 February 2015 # Springer Science+Business Media New York 2015
Abstract The mountain pine beetle, Dendroctonus ponderosae, depends on two fungi, Grosmannia clavigera and Ophiostoma montium, to augment a nutrient-poor woody food resource. Because the two fungi exert differential effects on the host beetle, temperature-driven differences in fungal growth and competition outcomes have a strong potential to influence host population dynamics. Weisolated fungi from beetles and wood from three locations in Montana and Utah, USA, and measured their growth rates and sporulation between 5 and 35 °C on artificial media. We also measured growth rates and percent resource capture for each fungus at 10, 15, 21, and 25 °C during inter- and intra-specific competition. G. clavigera excelled at resource capture at most temperatures. Its optimal growth temperature occurs around 20 °C while that of O. montium occurs near 30 °C. There was no effect of collection site on growth or sporulation; however, O. montium exhibited greater variability in response to temperature than did G. clavigera. Sporulation of G. clavigera was greatest at 30 °C while O. montium sporulated at low levels across all temperatures. During competition experiments, G. clavigera captured more resources than O. montium at most temperatures and captured a greater percentage of resources at a greater rate during inter-specific competition than during intra-specific competition. In contrast, O. montium captured a greater percentage of resources during intraspecific competition. These results demonstrate that temperature can differentially affect growth, sporulation, and resource capture of the two symbionts, indicating that it may be an important factor influencing the composition and dynamics of the symbiosis.
M. L. Moore : D. L. Six (*) Department of Ecosystem and Conservation Sciences, The University of Montana, Missoula, MT 59812, USA e-mail: [email protected]
Keywords Bark beetle . Symbiosis . Dendroctonus ponderosae . Grosmannia clavigera . Ophiostoma montium
Introduction The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) (Coleoptera: Curculionidae, Scolytinae) is a bark beetle indigenous to western North America. This insect is capable of killing a wide range of hosts in Pinus [38] and can cause the mortality of millions of trees during outbreaks. In recent years, damage caused by MPB has increased dramatically due to extensive outbreaks exacerbated by climate change, fire suppression, and logging practices [24, 28]. The beetle excels in the forest environment due, in part, to its participation in symbioses with two fungi, Grosmannia clavigera (Rob. & Jeff.) Zipfel, Z.W. de Beer & M.J. Wingfield and Ophiostoma montium (Rumbold) Arx, which are consistently associated with the beetle across its geographic range [18, 26, 34, 37]. MPB feeds on phloem, which is a poor nutrient resource relative to the needs of the insect [6, 32]. G. clavigera and O. montium provide nutritional supplementation by concentrating nitrogen [4, 6, 11, 15] and producing sterols [4]. Beetle larvae feed on the fungi along with phloem throughout their development [1, 6]. After completing development, the larvae create chambers in the phloem where they pupate and eclose to the adult form [32]. New adults feed on fungal spore layers that form on the walls of the pupal chamber [32]. As the beetles feed, they pack mycangia, specialized fungus transport structures in the exoskeleton, with spores that are then carried to the next tree and the next generation of beetles [9, 37]. Feeding on fungi has significant benefits for the beetles. Larvae that feed on phloem containing symbiotic fungi attain
M. L. Moore, D. L. Six
a larger adult body size [6, 32] which is positively correlated with fecundity [19]. They also have higher survival rates; larvae that feed on phloem alone seldom survive [37]. In addition, it appears that feeding on spores by new adults is required for reproduction [32]. While both fungi are able to support beetle growth and development in trees, they differ in the degree of benefit they confer [6, 11, 32]. G. clavigera concentrates nitrogen better than O. montium [11] which may account for observations that beetles developing with G. clavigera are bigger and have higher productivity and survival rates than those developing with O. montium [6, 15, 32]. Because differential nutrient supplementation by the two fungi translates directly to fitness effects on beetle productivity, the relative prevalence of the two fungi is predicted to influence MPB population dynamics. MPB must overwhelm tree defenses to colonize a tree and for the mycangial fungi to establish. To accomplish this, the beetles initiate a mass attack using a complex attractantantiattractant pheromone system that results in an even spacing of attacks on the tree bole [23]. This also produces a regular pattern of inoculation of the fungal associates within the tree. As the fungi grow out from their respective inoculation points, they eventually encounter other fungal individuals, either hetero- or con-specifics, growing from nearby beetle entry points. The relative prevalence of each species of fungus inoculated into the tree depends upon the proportion of the two symbionts being carried by attacking beetles that, in turn, is determined by temperature [34]. Once in the tree, rates of MPB development and fungal growth are directly controlled by temperature [2, 3, 5, 16]. Because both fungi are introduced into the tree at the same time, are limited to colonizing a finite phloem and sapwood resource, and ultimately must gain access to dispersing beetles for their spores to be transported to a new host tree, the two are expected to be in strong competition. Observational and experimental evidence in vitro, and in vivo in standing naturally attacked trees, indicates that the two fungi associated with MPB exhibit exploitation competition [6–8] where the outcome is determined solely by how efficiently or rapidly each species captures resources. Competitive interactions are thought to be destabilizing to multi-partite mutualisms and are predicted to result in the loss of inferior competitors over time [13]. However, the three-way symbiosis between MPB and its two fungi has remained remarkably stable over long periods of evolutionary time [30, 33]. Coexistence of competing organisms can occur when competitors respond differently to environmental conditions such that no one species is favored at all times [10]. Only two studies have directly examined how environmental factors may affect competition between the two fungi associated with MPB. In one study, conducted in vitro with the fungi grown at one temperature but at various water potentials mimicking those that occur in dying trees over time, both species
generally exhibited reduced growth rates and percent resource capture when grown with the other [8]. However, the growth rate of G. clavigera increased when it was grown in close proximity to O. montium at low water potentials. This indicates that under certain conditions, some fungal partners may be able to use volatiles, by-products, or enzymes diffusing from other partners to their advantage [8]. In the other study, the relative ability of the two fungi to capture space in naturally attacked trees was found to vary seasonally and depended upon the timing of their introduction to the host tree [7]. Together, these studies suggest that the fungi compete and capture resources in a manner highly dependent upon environmental conditions. Several studies have examined the growth rate responses of the two fungi at different temperatures. These studies found that G. clavigera is able to grow at lower temperatures than O. montium, while O. montium can grow at warmer temperatures than G. clavigera [25, 31, 36]. This may explain the shifting prevalence of the two fungi that has been observed over time in naturally attacked trees [1, 8]. Adams and Six [1] observed that G. clavigera was more often isolated from phloem adjacent to third and fourth instar larvae developing during spring and early summer when conditions were relatively cool, while O. montium was isolated more often from phloem adjacent to pupae, teneral adults, and eggs which develop in mid-to-late summer when temperatures are warmer. Temperature has also been correlated with the relative prevalence of the two fungi carried by dispersing beetles [34]. These studies collectively indicate that temperature determines the relative proportion of each fungus in a population and that this proportion shifts constantly over time. The effects of temperature on symbiont prevalence have important implications for both fungal and beetle fitness. For example, at sites that are generally cool, or at times of the year when conditions are overall cooler, G. clavigera would be expected to be at an advantage and capture the most resources within the tree. In contrast, at sites where conditions are generally warm, or during times of the year when conditions are warmer, O. montium would be expected to dominate. At sites where conditions fluctuate greatly, the two fungi would be expected to capture resources at different rates at different times of the year, with highly variable outcomes in total resources captured over time. Such variability in resource capture by the fungi should translate directly to effects on beetle fitness through differential nutrient availability during larval development. For example, an increase in the prevalence of O. montium during larval development should result in the production of more small adult female beetles [7] and a subsequent reduction in fecundity. While this initially may appear detrimental to MPB, a redundancy of nutritional symbionts, even if one is inferior to the other, may be critical to MPB persistence under fluctuating environmental conditions. By having more than one symbiont, each with different
Effects of Temperature on Mountain Pine Beetle Fungal Symbionts
temperature tolerances, MPB larvae may minimize the likelihood of being caught without a symbiont as conditions vary through time and space [34]. Differential responses to temperature may also support coexistence of the fungi over time by never allowing any one fungus to move to fixation with the host [2]. Our aim was to better understand the effects of temperature on the fungi associated with MPB. Past studies described temperature effects on growth rates of these fungi using very small numbers of isolates [25, 31, 36] and no studies have quantified how temperature affects sporulation or competition. In this study, we assessed the effects of temperature on the primary fitness parameters of the two fungi (growth rate, sporulation, and ability to compete with hetero- and con-specifics) in vitro. We included a large number of isolates from several locations in order to more accurately estimate growth rates and to capture the range of variability in responses to temperature within each species.
Methods Fungal Isolates A total of 88 isolates (50G. clavigera, 38 O. montium) were isolated from beetles and wood from two locations in MT and one location in UT, USA. Sites in MT were Vipond Park (45° 42.31′ N, 112° 55.54′ W, elevation 2438 m) located in the Beaverhead National Forest, and Lubrecht Experimental Forest (46° 53.30′ N, 113° 26.03′ W, elevation 1263 m) located in Missoula County. The UT site, Stump Hollow (41° 57.34′ N, 111° 31.47′ W, elevation 2136 m), was located in the UintaWasatch-Cache National Forest. Multiple sites were included to allow us to better describe the variability within each species, and also to detect if population sub-structuring occurred among fungi from geographically distant locations. All isolates were obtained either from dissections of MPB mycangia using methods described by Six and Paine [31], or from phloem removed from MPB galleries in trees using a sterile cork borer. Pure fungal isolates were obtained using single spore isolations and then incubated at room temperature on 2 % malt extract agar (MEA) enriched with sterile pine twig cuttings to encourage sporulation. Isolates were identified using cultural characteristics and morphology. Effects of Temperature on Growth Rates of G. Clavigera and O. Montium The 88 isolates were used to determine the growth rates of the fungi at various temperatures and to determine their upper and lower limits for growth. The isolates were grown for 5 days on 2 % MEA prior to use in each experiment to ensure that the cultures were in an active growth phase. On the fifth day, a 13-
mm2 circular plug of actively growing mycelium was extracted from the leading edge of the culture and placed mycelium side down onto the center of a 100-mm diameter Petri dish containing 2 % MEA. The inoculated plates were incubated for 14 days at 5, 10, 15, 21, 25, 30, and 35 °C. Each treatment was replicated three times for each isolate. At the same time each day, beginning on the second day, the area colonized by each fungus was traced on the bottom of the Petri dish. On the tenth day, the bottoms of the Petri dishes were photographed with a digital camera and photos uploaded onto a computer where the area colonized by each fungus per day was measured using ImageJ v.1.43u (ImageJ, Bethesda, MD). The cultures were held for an additional 4 days to assess sporulation (see below). Data Analyses Area measurements were regressed by time since inoculation and slopes fit by regression analysis. The slopes of the regression lines for each replicate were averaged for each isolate at each temperature, resulting in an average absolute growth rate for each. The mean, range, and standard error of the growth rate for each species at each temperature were determined. A logarithmic transformation was used to obtain relative growth rate estimates [21]. Mixed model analysis of variance (ANOVA, maximum likelihood method) with isolate as a random variable was used to test for the fixed effects of temperature, species, and site, and their interactions on relative growth rate. Significant F-tests for all interactions were followed by Tukey-Kramer’s honestly significant different (HSD) tests. For all tests, here and elsewhere, α was set at P
FUNGAL MICROBIOLOGY
Effects of Temperature on Growth, Sporulation, and Competition of Mountain Pine Beetle Fungal Symbionts Melissa L. Moore & Diana L. Six
Received: 30 December 2014 / Accepted: 26 February 2015 # Springer Science+Business Media New York 2015
Abstract The mountain pine beetle, Dendroctonus ponderosae, depends on two fungi, Grosmannia clavigera and Ophiostoma montium, to augment a nutrient-poor woody food resource. Because the two fungi exert differential effects on the host beetle, temperature-driven differences in fungal growth and competition outcomes have a strong potential to influence host population dynamics. Weisolated fungi from beetles and wood from three locations in Montana and Utah, USA, and measured their growth rates and sporulation between 5 and 35 °C on artificial media. We also measured growth rates and percent resource capture for each fungus at 10, 15, 21, and 25 °C during inter- and intra-specific competition. G. clavigera excelled at resource capture at most temperatures. Its optimal growth temperature occurs around 20 °C while that of O. montium occurs near 30 °C. There was no effect of collection site on growth or sporulation; however, O. montium exhibited greater variability in response to temperature than did G. clavigera. Sporulation of G. clavigera was greatest at 30 °C while O. montium sporulated at low levels across all temperatures. During competition experiments, G. clavigera captured more resources than O. montium at most temperatures and captured a greater percentage of resources at a greater rate during inter-specific competition than during intra-specific competition. In contrast, O. montium captured a greater percentage of resources during intraspecific competition. These results demonstrate that temperature can differentially affect growth, sporulation, and resource capture of the two symbionts, indicating that it may be an important factor influencing the composition and dynamics of the symbiosis.
M. L. Moore : D. L. Six (*) Department of Ecosystem and Conservation Sciences, The University of Montana, Missoula, MT 59812, USA e-mail: [email protected]
Keywords Bark beetle . Symbiosis . Dendroctonus ponderosae . Grosmannia clavigera . Ophiostoma montium
Introduction The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) (Coleoptera: Curculionidae, Scolytinae) is a bark beetle indigenous to western North America. This insect is capable of killing a wide range of hosts in Pinus [38] and can cause the mortality of millions of trees during outbreaks. In recent years, damage caused by MPB has increased dramatically due to extensive outbreaks exacerbated by climate change, fire suppression, and logging practices [24, 28]. The beetle excels in the forest environment due, in part, to its participation in symbioses with two fungi, Grosmannia clavigera (Rob. & Jeff.) Zipfel, Z.W. de Beer & M.J. Wingfield and Ophiostoma montium (Rumbold) Arx, which are consistently associated with the beetle across its geographic range [18, 26, 34, 37]. MPB feeds on phloem, which is a poor nutrient resource relative to the needs of the insect [6, 32]. G. clavigera and O. montium provide nutritional supplementation by concentrating nitrogen [4, 6, 11, 15] and producing sterols [4]. Beetle larvae feed on the fungi along with phloem throughout their development [1, 6]. After completing development, the larvae create chambers in the phloem where they pupate and eclose to the adult form [32]. New adults feed on fungal spore layers that form on the walls of the pupal chamber [32]. As the beetles feed, they pack mycangia, specialized fungus transport structures in the exoskeleton, with spores that are then carried to the next tree and the next generation of beetles [9, 37]. Feeding on fungi has significant benefits for the beetles. Larvae that feed on phloem containing symbiotic fungi attain
M. L. Moore, D. L. Six
a larger adult body size [6, 32] which is positively correlated with fecundity [19]. They also have higher survival rates; larvae that feed on phloem alone seldom survive [37]. In addition, it appears that feeding on spores by new adults is required for reproduction [32]. While both fungi are able to support beetle growth and development in trees, they differ in the degree of benefit they confer [6, 11, 32]. G. clavigera concentrates nitrogen better than O. montium [11] which may account for observations that beetles developing with G. clavigera are bigger and have higher productivity and survival rates than those developing with O. montium [6, 15, 32]. Because differential nutrient supplementation by the two fungi translates directly to fitness effects on beetle productivity, the relative prevalence of the two fungi is predicted to influence MPB population dynamics. MPB must overwhelm tree defenses to colonize a tree and for the mycangial fungi to establish. To accomplish this, the beetles initiate a mass attack using a complex attractantantiattractant pheromone system that results in an even spacing of attacks on the tree bole [23]. This also produces a regular pattern of inoculation of the fungal associates within the tree. As the fungi grow out from their respective inoculation points, they eventually encounter other fungal individuals, either hetero- or con-specifics, growing from nearby beetle entry points. The relative prevalence of each species of fungus inoculated into the tree depends upon the proportion of the two symbionts being carried by attacking beetles that, in turn, is determined by temperature [34]. Once in the tree, rates of MPB development and fungal growth are directly controlled by temperature [2, 3, 5, 16]. Because both fungi are introduced into the tree at the same time, are limited to colonizing a finite phloem and sapwood resource, and ultimately must gain access to dispersing beetles for their spores to be transported to a new host tree, the two are expected to be in strong competition. Observational and experimental evidence in vitro, and in vivo in standing naturally attacked trees, indicates that the two fungi associated with MPB exhibit exploitation competition [6–8] where the outcome is determined solely by how efficiently or rapidly each species captures resources. Competitive interactions are thought to be destabilizing to multi-partite mutualisms and are predicted to result in the loss of inferior competitors over time [13]. However, the three-way symbiosis between MPB and its two fungi has remained remarkably stable over long periods of evolutionary time [30, 33]. Coexistence of competing organisms can occur when competitors respond differently to environmental conditions such that no one species is favored at all times [10]. Only two studies have directly examined how environmental factors may affect competition between the two fungi associated with MPB. In one study, conducted in vitro with the fungi grown at one temperature but at various water potentials mimicking those that occur in dying trees over time, both species
generally exhibited reduced growth rates and percent resource capture when grown with the other [8]. However, the growth rate of G. clavigera increased when it was grown in close proximity to O. montium at low water potentials. This indicates that under certain conditions, some fungal partners may be able to use volatiles, by-products, or enzymes diffusing from other partners to their advantage [8]. In the other study, the relative ability of the two fungi to capture space in naturally attacked trees was found to vary seasonally and depended upon the timing of their introduction to the host tree [7]. Together, these studies suggest that the fungi compete and capture resources in a manner highly dependent upon environmental conditions. Several studies have examined the growth rate responses of the two fungi at different temperatures. These studies found that G. clavigera is able to grow at lower temperatures than O. montium, while O. montium can grow at warmer temperatures than G. clavigera [25, 31, 36]. This may explain the shifting prevalence of the two fungi that has been observed over time in naturally attacked trees [1, 8]. Adams and Six [1] observed that G. clavigera was more often isolated from phloem adjacent to third and fourth instar larvae developing during spring and early summer when conditions were relatively cool, while O. montium was isolated more often from phloem adjacent to pupae, teneral adults, and eggs which develop in mid-to-late summer when temperatures are warmer. Temperature has also been correlated with the relative prevalence of the two fungi carried by dispersing beetles [34]. These studies collectively indicate that temperature determines the relative proportion of each fungus in a population and that this proportion shifts constantly over time. The effects of temperature on symbiont prevalence have important implications for both fungal and beetle fitness. For example, at sites that are generally cool, or at times of the year when conditions are overall cooler, G. clavigera would be expected to be at an advantage and capture the most resources within the tree. In contrast, at sites where conditions are generally warm, or during times of the year when conditions are warmer, O. montium would be expected to dominate. At sites where conditions fluctuate greatly, the two fungi would be expected to capture resources at different rates at different times of the year, with highly variable outcomes in total resources captured over time. Such variability in resource capture by the fungi should translate directly to effects on beetle fitness through differential nutrient availability during larval development. For example, an increase in the prevalence of O. montium during larval development should result in the production of more small adult female beetles [7] and a subsequent reduction in fecundity. While this initially may appear detrimental to MPB, a redundancy of nutritional symbionts, even if one is inferior to the other, may be critical to MPB persistence under fluctuating environmental conditions. By having more than one symbiont, each with different
Effects of Temperature on Mountain Pine Beetle Fungal Symbionts
temperature tolerances, MPB larvae may minimize the likelihood of being caught without a symbiont as conditions vary through time and space [34]. Differential responses to temperature may also support coexistence of the fungi over time by never allowing any one fungus to move to fixation with the host [2]. Our aim was to better understand the effects of temperature on the fungi associated with MPB. Past studies described temperature effects on growth rates of these fungi using very small numbers of isolates [25, 31, 36] and no studies have quantified how temperature affects sporulation or competition. In this study, we assessed the effects of temperature on the primary fitness parameters of the two fungi (growth rate, sporulation, and ability to compete with hetero- and con-specifics) in vitro. We included a large number of isolates from several locations in order to more accurately estimate growth rates and to capture the range of variability in responses to temperature within each species.
Methods Fungal Isolates A total of 88 isolates (50G. clavigera, 38 O. montium) were isolated from beetles and wood from two locations in MT and one location in UT, USA. Sites in MT were Vipond Park (45° 42.31′ N, 112° 55.54′ W, elevation 2438 m) located in the Beaverhead National Forest, and Lubrecht Experimental Forest (46° 53.30′ N, 113° 26.03′ W, elevation 1263 m) located in Missoula County. The UT site, Stump Hollow (41° 57.34′ N, 111° 31.47′ W, elevation 2136 m), was located in the UintaWasatch-Cache National Forest. Multiple sites were included to allow us to better describe the variability within each species, and also to detect if population sub-structuring occurred among fungi from geographically distant locations. All isolates were obtained either from dissections of MPB mycangia using methods described by Six and Paine [31], or from phloem removed from MPB galleries in trees using a sterile cork borer. Pure fungal isolates were obtained using single spore isolations and then incubated at room temperature on 2 % malt extract agar (MEA) enriched with sterile pine twig cuttings to encourage sporulation. Isolates were identified using cultural characteristics and morphology. Effects of Temperature on Growth Rates of G. Clavigera and O. Montium The 88 isolates were used to determine the growth rates of the fungi at various temperatures and to determine their upper and lower limits for growth. The isolates were grown for 5 days on 2 % MEA prior to use in each experiment to ensure that the cultures were in an active growth phase. On the fifth day, a 13-
mm2 circular plug of actively growing mycelium was extracted from the leading edge of the culture and placed mycelium side down onto the center of a 100-mm diameter Petri dish containing 2 % MEA. The inoculated plates were incubated for 14 days at 5, 10, 15, 21, 25, 30, and 35 °C. Each treatment was replicated three times for each isolate. At the same time each day, beginning on the second day, the area colonized by each fungus was traced on the bottom of the Petri dish. On the tenth day, the bottoms of the Petri dishes were photographed with a digital camera and photos uploaded onto a computer where the area colonized by each fungus per day was measured using ImageJ v.1.43u (ImageJ, Bethesda, MD). The cultures were held for an additional 4 days to assess sporulation (see below). Data Analyses Area measurements were regressed by time since inoculation and slopes fit by regression analysis. The slopes of the regression lines for each replicate were averaged for each isolate at each temperature, resulting in an average absolute growth rate for each. The mean, range, and standard error of the growth rate for each species at each temperature were determined. A logarithmic transformation was used to obtain relative growth rate estimates [21]. Mixed model analysis of variance (ANOVA, maximum likelihood method) with isolate as a random variable was used to test for the fixed effects of temperature, species, and site, and their interactions on relative growth rate. Significant F-tests for all interactions were followed by Tukey-Kramer’s honestly significant different (HSD) tests. For all tests, here and elsewhere, α was set at P
