Laboratory and Field Evaluation of the Entomopathogenic Fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes) for Population Management of Spruce Beetle, Dendroctonus rufipennis (Coleoptera: Scolytinae), in Felled Trees and Factors Limiting Pathogen Success

Laboratory and Field Evaluation of the Entomopathogenic Fungus Beauveria bassiana... Abstract An isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) was tested for its ability to reduce survival and reproduction of spruce beetle, Dendroctonus rufipennis (Kirby) (Coleoptera: Scolytinae), under laboratory and field conditions. Conidial suspension applied directly to adults or to filter papers that adults contacted had a median survival time of 3–4 d in laboratory assays and beetles died more rapidly when exposed to conidial suspension than when treated with surfactant solution only. In the field, conidial suspension was applied to the surface of felled and pheromone-baited Engelmann spruce (Picea engelmannii) trees using a backpack sprayer. Mortality of colonizing parent beetles (F0), reproduction (abundance of F1 offspring in logs), and emergence of F1 beetles from logs was compared between treated and nontreated logs. Application of spore suspension increased mortality of F0 adults by 36% on average. Total F1 reproduction was reduced by 17% and emergence from logs was reduced by 13% in treated logs, but considerable variability in reproduction and emergence was observed. Viable spores were re-isolated from treated logs up to 90 d after application, indicating that spores are capable of long-term persistence on the tree bole microhabitat. Subsequent in vitro tests revealed that temperatures below 15°C and exposure to spruce monoterpenes likely limit performance of B. bassiana under field conditions, but exposure to low-intensity light or interactions with spruce beetle symbiotic fungi were not strongly inhibitory. It is concluded that matching environmental tolerances of biocontrol fungi to field conditions can likely improve their usefulness for control of spruce beetle in windthrown trees. bark beetle, Beauvaria, Curculionidae, forest entomology, microbial control In the western United States, the spruce beetle, Dendroctonus rufipennis (Kirby) (Coleoptera: Scolytinae), is associated with death or decline of at least 14M Engelmann spruce (Picea engelmanii Parry ex Engelm.) trees since 1995 (Holsten et al. 1999, Wittwer 2000, Jenkins et al. 2014, Harris 2016). Endemic spruce beetle populations infest primarily weaken or stressed hosts and often kill host trees over several years through multiple re-infestations (Massey and Wygant 1954); however, high-density spruce beetle populations (‘epidemics’) preferentially attack healthy, mature spruce trees and can cause mortality within a single growing season. The transition from endemic to epidemic population phases can occur when large quantities of ‘windthrown’ trees become available to endemic populations due to blowdown events (Dyer and Taylor 1971, Schmid 1981, Raffa et al. 2008, Safranyik 2011). Windthrow occurring during winter or spring may remain green throughout the subsequent growing season and can be colonized by D. rufipennis. Colonization of windthrown spruce by beetles facilitates rapid population buildup as host material lacks the ability to mount an induced defensive response in reaction to infestation; some reports indicate that brood production of D. rufipennis in windthrown trees can exceed that in live hosts by 200–300% (Hebertson and Jenkins 2007), which may facilitate population growth beyond endemic thresholds. Consequently, methods are needed for controlling beetle population buildup in green, undefended windthrown trees. Although various approaches to beetle population control including silvicultural treatments (Sartwell and Stevens 1975, Fettig et al. 2007), chemical suppression techniques (Fettig et al. 2006), and integrated pest management tactics can reduce probability of infestation (Hansen et al. 2017), comparatively few biological methods are available. However, ‘biopesticide’ plant protectants including microorganisms are easier to license than traditional chemical means and less environmentally hazardous than commonly recommended chemical applications for beetle control such as carbaryl and pyrethroids (e.g., Bridges 2000, Ribera et al. 2001, Rohr et al. 2003). In addition, some biological alternatives to chemical application have a higher degree of host specificity and relatively shorter environmental half-life (Strasser et al. 2000, Ownley et al. 2004, Zimmerman 2007). These potential advantages have led to renewed interest in the application of biological control agents, including entomopathogenic fungi, for population control of beetles. Here, an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.-Criv.) Vuill. (1912) (Deuteromycotina: Hyphomycetes) is tested for its ability to reduce survival and population growth of D. rufipennis in P. engelmannii in the central Rocky Mountains. The use of B. bassiana and other entomopathogenic fungi for population management for pest insects has been an active area of research for decades (Feng et al. 1994, Hajek and St. Leger 1994, Meyling and Eilenberg 2007). Beauveria bassiana is an effective biological control agent against a variety of insect pests, including agricultural and stored products pests (e.g., Butt et al. 1994, Moino et al. 1998, Padin et al. 2002, Hirsch and Reineke 2014, Rondot and Reineke 2017), and B. bassiana has been tested extensively against some angiosperm-infesting bark beetles (e.g., Doberski and Tribe 1980, Doberski 1981) and other curculionids (Kreutz et al. 2004a, b; Batta 2007). Collectively, these reports indicate that certain isolates or formulations of B. bassiana may be generally effective as a biological control against wood-boring beetles. However, relatively few studies have tested the potential for B. bassiana to control populations of conifer-infesting Dendroctonus bark beetles in the field (Pabst and Sikorowski 1980, Hunt et al. 1984, Sevim et al. 2009, Zhang et al. 2011), though the fungus occurs frequently in Dendroctonus populations and habitats (Moore 1971, Ormond et al. 2010, Yao et al. 2012). Augmentation of natural entomopathogen populations may be a useful method for reducing spruce beetle reproduction in windthrown trees. Accordingly, our objectives were 1) to evaluate pathogenicity for a selected isolate in laboratory bioassays, 2) test the isolate under field conditions as a spray formulation for protection of spruce logs challenged with pheromone baits, and 3) assess environmental factors that may limit the success of field applications. Materials and Methods Isolation and Identification of Test Organisms Isolates were collected from serial dilutions of surface washes of bark, phloem, or duff taken from ponderosa (Pinus ponderosa Douglas ex C. Lawson), lodgepole pine (Pinus contorta Douglas), and Engelmann spruce; surface washes were also made from Dendroctonus ponderosae Hopkins (Coleoptera: Scolytinae) and D. rufipennis adults. Up to 10 g of material for serial dilution was surface washed in 100 ml of 0.01% Silwet L77 (Helena Chemical Company, Collierville, TN) on a table shaker for 15 min at 100 r.p.m. Serial dilutions of surface washes were spread onto potato dextrose agar amended with 0.05% dodine and 0.003% gentamicin (modified from Rangel et al. 2010) and incubated at 25°C in the dark for up to 15 d. For colonies sporulating on dodine agar and with colony morphology consistent with descriptions for Beuavaria bassiana, single colony forming units were inoculated onto Sabouraud Dextrose Agar (SDA) and allowed to sporulate for entomopathogenecity tests. For select isolates, entomopathogenecity was confirmed in a preliminary laboratory bioassay. D. rufipennis adults were captured using Lindgren funnel traps baited with pheromone lures containing frontalin, 1-methylcyclohex-2-en-1-ol (MCOL), and a proprietary blend of host tree volatiles (monoterpenes; Synergy Semiochemicals, Victoria, British Columbia, Canada) and placed individually into 6 cm diameter Petri dishes. A sterile 10-µl inoculating loop was wetted in 0.01% Silwet L77 and pressed into sporulated colonies to collect spores; spores were transferred to beetles by contact with the inoculating loop and beetles were checked daily for evidence of sporulation during a 7-d period. If sporulation was noted, single colonies were re-isolated onto potato dodine agar and transferred to SDA and again allowed to sporulate. Conidial spores were harvested into cryovials containing 30% glycerol and stored at −30°C until used. In total, 30 pure cultures obtained in this way were selected for genotyping of the 28S gene (318 bp) at a commercial facility (MIDI Labs, Newark, DE), and all were consistent with B. Bassiana sensu lato (>99% sequence similarity to B. bassiana M9344; GenBank AB576868)—since recent studies indicate cryptic speciation in the Beauvaria genus (Rehner et al. 2011), we do not assign a molecular characterization beyond the broad sense. A single putative B. bassiana isolate was selected for further laboratory and field testing on the basis of abundant conidia production and observed pathogenicity to D. rufipennis. Spore Production for Bioassays Spore preparations for laboratory and field testing were produced using solid substrate culture. Culture substrate was dehulled barley wetted with a salt solution (MgSO4: 0.5 g/liter, NaCl: 0.1 g/liter, KCl: 0.1 g/liter, FeCl3: 0.02 g/liter, (NH4)2HPO4: 5g/liter, and yeast extract 1.0 g/liter) to a final moisture content of 55% (substrate is moist, but there is no freestanding water). Salt solution was amended to the substrate and periodically mixed for 1 h to ensure complete absorption of salts into barley substrate. The mixture was autoclaved and the selected isolate was inoculated into the mixture by aseptic transfer of yeast extract broth containing the selected B. bassiana isolate at a ratio of 10 ml inoculum broth/100 g substrate. Solid cultures of ~500 g were incubated in bags aerated with sterile air at a constant flow of 500 ml/min. Sporulation and subsequent spore harvest occurred 7–10 d following aeration. To harvest spores, cultures were transferred to perforated aluminum trays and placed in a chamber with a constant flow of dehumidified, filtered air. Cultures dried within 48 h and were processed for spore recovery by sieving through 250-micron mesh. Spore concentration in dry cultures ranged from 5 × 109 to 4 × 1010 spores/g. Dry culture was milled to produce spore powder. Conidial viability from milled and sieved culture was determined by germination test. The ratio of germinated to ungerminated conidia was determined by microscope count of conidia incubated on water agar for 16 h. Conidia were considered germinated if a visible germ tube with length greater than the conidia diameter was observable, and approximately 200 conidia were counted from aggregate sieved culture (Milner et al. 1991). The viability of conidia prepared from solid substrate culture was determined to be >90%. Conidia powder diluted into a surfactant solution was used for laboratory bioassays. At a concentration of 1 mg conidia powder/1 ml 0.01% Silwet L77, the concentration of colony forming units (CFUs) was 2.4 × 108 CFU/ml. For field trials, conidia powder was mixed 1:5 in a clay-based carrier and the formulation was diluted into water with 0.01% Silwet L77 to a final concentration of 1.5 × 109 CFU/ml for spray treatment. Pathogenicity of B. bassiana to D. rufipennis in Laboratory Bioassays Using conidia produced from solid substrate culture, two laboratory assays were performed to evaluate the efficacy of the selected B. bassiana isolate for causing mortality in D. rufipennis: 1) application of conidial suspension, applied to individual beetles, and 2) application of conidial suspension to filter papers on which beetles were confined for the duration of the observation period. Both tests took place in the dark at an ambient temperature of 23°C and relative humidity ~30%. In the first test, forty new adult beetles were excavated from infested P. engelmannii material and placed individually into 3-cm diameter Petri dishes. A B. bassiana conidial suspension (1.8 × 109 CFU/ml) was directly applied to the ventral surface of the thorax and abdomen of 20 beetles in 20-µl aliquots using a pipette (treatment group), and the remaining 20 beetles were treated with 20-µl aliquots of sterile distilled water as a control. Beetles were checked daily for mortality, and the number of days until death was recorded for each individual. In the second test, each replicate consisted of 10 test beetles (as above, freshly excavated from infested P. engelmannii material) placed in 6-cm diameter Petri dishes containing filter papers (Whatman Grade 6, 4.25-cm diameter). For half the replicates (n = 10), 1 ml of B. bassiana conidial suspension (1.8 × 109 CFU/ml) was applied to the filter paper (treatment group); for the remaining replicates (n = 10), 1 ml of sterile distilled water was applied to filter papers as a control. As in the first test, cumulative beetle mortality within each Petri dish was recorded until all beetles had died. Field test of B. bassiana formulation on D. rufipennis performance The field test took place in northern Wyoming in the vicinity of Togwotee Pass (2,945 m a.s.l., approximate location: 43.825704°N, 110.214377°W). In the study area, spruce beetle population pressure was significant, with ongoing high-density D. rufipennis populations causing extensive Engelmann spruce mortality. Stands of Engelmann spruce were selected for the field test based on recent observations of tree mortality from D. rufipennis activity. Post experiment spruce beetle risk rating ranged from low-moderate to moderate based on the criteria of Schmid and Frye (1977). In total, 36 individual spruce trees were tagged for study and selected for felling; mean tree diameter at breast height of study trees was 36 ± 0.4 cm SE. During June 2016, trees were felled at 0.5 m from the ground and baited with spruce beetle pheromone lure containing frontalin, MCOL, and a proprietary blend of host tree volatiles (monoterpenes; Synergy Semiochemicals). Baits were placed in a shaded portion of the center of the bole of felled trees. Conidial suspension treatment was applied to half (n = 18) of the felled trees, and the remaining trees (n = 18) were left as nontreated controls. Conidial suspension was applied to downed trees using a Solo model 433 motorized backpack equipped with a ‘dual Y’ fan nozzle. Total application volume ranged from approximately 4–5 liters per tree, and the suspension was applied to all visible tree surfaces, with careful attention to application where tree boles contacted the ground; the ground surface near each treatment tree was treated as well. In September 2016, all study trees were cut into three 40-cm logs starting from the base; remaining tree material (upper bole and crown) was bucked and piled. Bark samples were collected from six treated trees using a 5-cm hole saw; samples were surface washed and serially diluted to estimate CFUs B. bassiana/cm2 bark. One of the three logs were randomly chosen for processing in the field to estimate mortality of colonizing F0 beetles, the remaining two logs were sealed at the ends with paraffin wax and returned to the laboratory for rearing. For logs chosen to quantify F0 mortality, three 40 × 8 cm strips (320 cm2) were cut from the bark using a bark grinder. All live and dead D. rufipennis adults and all larvae were counted within each strip and counts were normalized to express percent mortality. Strips counted in this way were treated as subsamples, and percent mortality values from strips were averaged for each log. Logs returned to the laboratory were used to determine both survival and emergence of F1 beetles. Logs were placed in rearing containers ventilated with 1 × 1-mm mesh and kept at 23°C. Logs accumulated ~800 degree-days (base 10°C) in the laboratory, after which point half the logs from each study tree (n = 18 treatment logs, and n = 18 control logs) were randomly selected for collection of F1 adults. Bark was removed from logs selected for collection of F1 adults using a draw knife, and all adult D. rufipennis were collected and counted. Beetle abundances were standardized to the surface area (beetles/dm2) of each log. Ips pilifrons (Swaine) (Coleoptera: Curculionidae) beetles also emerged from logs in notable quantities, so were collected and quantified as well . The remaining logs (n = 18 treatment logs, and n = 18 control logs) were placed outside at Waverly Experimental Ranch in northern Colorado (Colorado State University) for winterization from November 2016 to February 2017. Logs were kept in rearing containers during winterization. After 95 d (February 2017), rearing containers containing logs were returned to the laboratory and placed at 23°C. Rearing containers were checked every 48 h for newly emerged adults. Only D. rufipennis and I. pilifrons, which emerged in significant abundances from logs and were also considered in statistical analyses, were collected and counted, and counts were standardized to the surface area (beetles/dm2) of each log. Collection of emerging F1 beetles continued until no further emergence was observed. Factors Limiting Growth of B. bassiana The B. bassiana isolate used in laboratory and field entomopathogenicity tests was also subjected to a panel of in vitro assays to address isolate responses to environmental and biotic factors hypothesized to limit efficacy in the field. Specifically, the response of the isolate to 1) variation in temperature, 2) variable photoperiods, 3) exposure to secondary chemicals in the spruce subcortical environment (monoterpenes), and 4) interactions with the spruce beetle symbiotic fungus Leptographium abietinum were tested. 1) Temperature. Three replicates of the B. bassiana isolate were grown on Petri dishes containing 20% MEA in dark incubation chambers maintained at 5, 10, 15, 20, 23, 25, 30, and 35°C. Radial growth of replicates was recorded daily for 15 d or until hyphal growth reached the end of dishes. 2) Photoperiod. Five replicates were of the B. bassiana isolate were grown on petri dishes containing 20% MEA at 23°C (the optimal temperature identified for growth) in incubation chambers maintained at four photoperiods; 0, 6, 12, or 24 h at a constant light intensity of 14 µmol/m2/s (Sylvania Supersaver Cool White F40CW/SS 34Watt). This is a low light intensity that is about 5% of that experienced in full sunlight. Radial growth of replicates was recorded daily for 15 d or until hyphal growth reached the end of dishes. 3) Monoterpenes. Five replicates of the B. bassiana isolate were grown in 20% MEA at 23°C amended with one of two monoterpenes found in the phloem of all Engelmann spruce tested thus far, (+)-α-pinene (98% purity, Sigma-Aldrich) and (+)-3-carene (>90% purity, Sigma-Aldrich), at three concentrations including 0.1, 1, and 5% (v/v), consistent with constitutive (0.1–1%) and induced (5%) monoterpene concentrations (Davis et al. 2018). Radial growth of replicates was recorded daily for 15 d or until hyphal growth reached the end of dishes. 4) Interactions with L. abietinum. A final test considered the ability of B. bassiana to compete with the spruce beetle symbiotic fungus Leptographium abietinum (Peck) M.J. Wingf.; the fungus is associated with nearly all spruce beetles tested (>95%) from across the range (Six and Bentz 2003) and may be able to prevent pathogenic or opportunistic fungi from occupying the subcortical gallery environment. To assess the potential for each fungus to capture and maintain resource area when co-occurring, 10 replicates of the B. bassiana isolate were competed against 10 unique isolates of L. abietinum (isolation protocol described in Davis et al. 2018) by inoculating each fungus equidistant onto Petri dishes containing 20% MEA, and trials occurred at 23°C in the dark. Isolates were allowed to grow for 20 d after which point they were scored by scanning dishes (Epson V600) and analyzing images using the software ImageJ (Schneider et al. 2012) to estimate the proportion (%) of each dish occupied by each fungus. Data Analysis In both laboratory assays, time-until-death following treatment (median survival time, MST) was analyzed using the Kaplan–Meier estimator (Kaplan and Meier 1958) to test for differences in the survival function due to treatment effects using a log-rank test approximated by a chi-square distribution and implemented in the R add-on package ‘survival’ (Therneau and Lumley 2017). In field trials, the response variables of F0 percent mortality, F1 survival in logs, and F1 emergence from logs were analyzed using a two-sample Student’s t-test to compare trees treated with spore suspension to nontreated control trees, with each log treated as an experimental replicate. In in vitro assays testing B. bassiana responses to varying environmental conditions, the response of radial growth rate (mm/d) was analyzed using one-way ANOVA to test the fixed effects of temperature (5, 10, 15, 20, 23, 25, 28, 30, and 35°C), photoperiod (0, 6, 12, and 24 h), and concentration (0.1, 1, and 5%) of monoterpenes. A two-sample Student’s t-test was used to compare the mean proportion of total Petri dish area occupied by B. bassiana and L. abietinum in resource competition tests. Data were checked for adherence to assumptions of normality and equal variances, and all response variables conformed to these assumptions. All parametric statistical tests were performed using the software JMP 12.0 (SAS Institute, Cary, NC) and a type I error rate of α = 0.05. Results Pathogenicity of B. bassiana to D. rufipennis in Laboratory Bioassays Direct application of spore suspension to D. rufipennis adults in the laboratory significantly impacted MST of beetles, and beetles treated with spore suspension died 20% more rapidly on average (MST: 4 d) than beetles treated only with distilled water (MST: 5 d; χ2= 4.113, df = 1, n = 40, P = 0.042; Fig. 1a). Similarly, application of spore suspension to filter papers contacted by D. rufipennis adults significantly reduced beetle survival time by 33% on average, with an MST of 43.0 d for beetles exposed to filter papers treated with spore suspension compared with an MST of 6 d for beetles exposed to filter papers treated with only water (χ2 = 72.878, df = 1, n = 200, P < 0.001; Fig. 1b). Fig. 1. View largeDownload slide Kaplan–Meier survivorship curves reflecting time-until-death for D. rufipennis in laboratory assays following a) direct application of spore suspension to beetles, or b) application of spore suspension to filter papers contacted by adult beetles. Solids lines and closed circles denote B. bassiana treatments, dashed lines, and open circles denote control treatments; bars show plus or minus on standard error of the mean. Fig. 1. View largeDownload slide Kaplan–Meier survivorship curves reflecting time-until-death for D. rufipennis in laboratory assays following a) direct application of spore suspension to beetles, or b) application of spore suspension to filter papers contacted by adult beetles. Solids lines and closed circles denote B. bassiana treatments, dashed lines, and open circles denote control treatments; bars show plus or minus on standard error of the mean. Field Test of B. bassiana Formulation on D. rufipennis Performance In total, 1,404 living and 978 dead D. rufipennis adults were counted from removed bark strips. Treatment with clay-based spore suspension reduced survival of adult D. rufipennis colonizing and ovipositing in baited host material by 36% on average, and this difference was statistically significant (t34 = 4.324; P < 0.0001; Fig. 2a). However, no evidence was found for reduced survival of larvae (t34 = 0.227; P < 0.821) resulting from application of spore suspension, and it was not possible to separate Ips from Dendroctonus larvae in field counts; thus, evaluation of spore suspension on larval survival in the field considers the sum of Dendroctonus and I. pilifrons larvae. Fig. 2. View largeDownload slide a) Relative mortality of colonizing adult (F0) D. rufipennis following entry into logs treated with a clay-based spore suspension of B. bassiana (treatment group) or nontreated control logs. b) Brood (abundance of F1 offspring) counted in logs, and (c) emergence of brood from logs. Open gray circles denote sample data, closed black circles denote sample means, and bars show one standard error. Asterisk denotes significant (P < 0.05) differences between treatment and control. Fig. 2. View largeDownload slide a) Relative mortality of colonizing adult (F0) D. rufipennis following entry into logs treated with a clay-based spore suspension of B. bassiana (treatment group) or nontreated control logs. b) Brood (abundance of F1 offspring) counted in logs, and (c) emergence of brood from logs. Open gray circles denote sample data, closed black circles denote sample means, and bars show one standard error. Asterisk denotes significant (P < 0.05) differences between treatment and control. In total, 3,989 D. rufipennis and 1,557 I. pilifrons brood were produced in logs peeled for quantification of reproduction (i.e., abundance of F1 offspring in logs prior to emergence). Mean D. rufipennis brood (F1) abundance in spruce logs was 3.4 beetles/dm2 in nontreated logs, and 2.7 beetles/dm2 in logs treated with spore suspension, but this difference was not statistically significant (t31 = −0.885, P = 0.382; Fig. 2b). Similarly, reproduction of I. pilifrons in spruce logs was not significantly affected by treatment with spores (t26 = 0.396, P = 0.695), and I. pilifrons abundance in nontreated logs was 1.3 beetles/dm2, whereas abundance in treated logs was 1.7 beetles/dm2. The sum of D. rufipennis adults and I. pilifrons adults was also not significantly affected by treatment of logs with spores (t26 = 0.075, P = 0.785). Mean emergence of D. rufipennis F1 brood from winterized spruce logs was nearly two orders of magnitude less than what was counted from peeled logs during evaluation of reproduction, with only 208 adult D. rufipennis emerging (~5% emergence). Although there was a 13% reduction in D. rufipennis emergence from treated versus nontreated logs, this result was not statistically significant (t35 = 0.017, P = 0.893; Fig. 2c), and no D. rufipennis brood emerged from 44% of logs (16 of 36 experimental units). Similarly, emergence of I. pilifrons (t35 = 0.233, P = 0.632) and the sum of D. rufipennis and I. pilifrons (t35 = −0.492, P = 0.625) were not affected by treatment of logs with spore suspension. Analysis of bark samples from treated logs indicated that immediately following initial treatment viable CFUs ranged from 5.3 × 106–2.3 × 107 viable CFU/cm2 bark (mean = 1.0 × 107); however, after 90 d in the field, viable CFUs/cm2 bark averaged 1.5 × 105. Factors Limiting Growth of B. bassiana 1) Temperature. Temperature had a considerable effect on variation in mean B. bassiana radial growth rates (F7, 16 = 15.528; P < 0.001). Growth rate was maximized at 23°C in the dark but was comparable across temperatures ranging from 20–30°C. There was a significant decline in growth rate at 15°C, and growth at 10°C was reduced by84% on average in comparison with growth at 15°C and by 94% on average in comparison with growth at 23°C. Mycelia failed to establish at 5 and 35°C, indicating the lower and upper bounds of growth for the isolate (Fig. 3a). 2) Photoperiod. There was no effect of photoperiod (0, 6, 12, or 24 h) on mycelial growth of B. bassiana at the light intensity tested (F3, 14 = 2.177; P = 0.013; Fig. 3b). 3) Monoterpenes. Inoculation into dishes amended with either monoterpene uniformly retarded B. bassiana growth (α-pinene test: F2, 12 = 331.790; P < 0.001; 3-carene test: F2, 12 = 7.657; P = 0.007); at constitutive concentrations (0.1–1%) the test isolate was still capable of growing at 70–80% of the rate of mycelia growing in the absence of monoterpenes. However, at concentrations that might be expected in defensively induced Engelmann spruce trees (5%), growth in media amended with α-pinene or 3-carene was completely suppressed or reduced by 83% on average, respectively (Fig. 3c). 4) Interactions with L. abietinum. The test isolate of B. bassiana appeared to be an equal competitor with L. abietinum in a resource limited environment, with neither species occupying a greater proportion of resource area on average (t9 = 0.249; P = 0.808; Fig. 3d). Fig. 3. View largeDownload slide Effects of multiple environmental conditions on the in vitro growth performance of B. bassiana test isolate, including a) temperature variability; b) photoperiod; c) exposure to monoterpenes; and d) interactions with the spruce beetle symbiotic fungus, L. abietinum. Lettering denotes Tukey’s HSD test, and bars show plus or minus one standard error of the mean. Aside from data summarized in (a), all tests were performed at 23°C. Fig. 3. View largeDownload slide Effects of multiple environmental conditions on the in vitro growth performance of B. bassiana test isolate, including a) temperature variability; b) photoperiod; c) exposure to monoterpenes; and d) interactions with the spruce beetle symbiotic fungus, L. abietinum. Lettering denotes Tukey’s HSD test, and bars show plus or minus one standard error of the mean. Aside from data summarized in (a), all tests were performed at 23°C. Discussion Laboratory tests indicated that the selected B. bassiana isolate was entomopathogenic; treatment with conidial suspension caused a decrease in median adult D. rufipennuis survival time of 21–40%, depending on the bioassay (Fig. 1a and b). The effect of treatment on reducing D. rufipennis MST was greater in the assay where beetles merely contacted treated surfaces (filter papers), which is the same mode of application used in treating downed trees. Although the tested B. bassiana isolate was effective at killing D. rufipennis adults contacting treated surfaces under laboratory conditions, this did not translate to field efficacy. In field tests, mortality of parent beetles (F0) colonizing highly attractive (pheromone-baited) felled trees after contact with surface treatments was significantly higher than in nontreated felled trees (Fig. 2a); however, this effect did not result in a reduction to F1 brood size (Fig. 2b) or emergence of brood from billets (Fig. 2c). Various environmental effects may limit the efficacy of B. bassiana in pest control applications and could potentially explain the disparity observed here between laboratory and field tests. For instance, ambient temperature and relative humidity interact to impact infection rates in larvae of the elm bark beetle, Scolytus scolytus (F.) (Coleoptera: Scolytinae) by B. bassiana (Doberski 1981), with fungal performance generally optimized at temperatures between 20 and 25°C. Exposure to ultraviolet light (particularly UV-B radiation) is also considered detrimental to the growth and persistence of the fungus (Inglis et al. 1995). These two factors are often reported as primary limitations to B. bassiana efficacy in the field. In the present study, environmental conditions over the duration of the field trial did not match optimal conditions determined by a posteriori laboratory assays. Daily temperature and humidity records summarized at a 4-km spatial resolution from June–August (2016), the temporal window during which experimental treatments and D. rufipennis flight and host colonization occurred in our study area, showed that mean temperature and relative humidity during field tests were approximately 13.5°C and 37%, respectively (PRISM Climate Group 2018). However, our in vitro experiments indicate that the optimal thermal range for the tested B. Bassiana isolate were between 20–30°C, with an 84% decline in mycelial growth when ambient temperatures decreased from 15 to 10°C (Fig. 3a). Exposure to all tested low-intensity photoperiods (6–24 h at 14 µmol/m2 min) had a weak-to-modest negative effect (9–17% reduction) on mycelial growth on average, but this was not statistically significant. In addition to suboptimal environmental conditions, Dendroctonus spp. may be low-quality hosts for B. bassiana. Hunt et al. (1984) determined that germination of conidia is suppressed on the cuticle of mountain pine beetle (D. ponderosae) due to nutrient deficiencies. The sclerotized cuticle lacks essential nutrients required for B. bassiana germination, although beetle hemolymph is a sufficient to induce extensive germination. Accordingly, adult beetles lacking entry points into the hemolymph may be somewhat resistant to colonization by the fungus, particularly if spores or new germinants are immediately exposed to tree secondary chemicals. B. bassiana infecting Dendroctonus spp. must contend with exposure to tree secondary compounds, especially, monoterpenes, which may permeate the subcortical egg-gallery environment and can inhibit growth of many fungal species (Hofstetter et al. 2005, Davis and Hofstetter 2012, Marei et al. 2012). Several studies have demonstrated that exposure to fungistatic compounds on the phylloplane can reduce efficacy of entomopathogenic fungi (e.g., Inyang et al. 1998, Poprawski and Jones 2001). Exposure to all tested concentrations (0.1, 1, and 5% v/v) of two monoterpenes commonly found in Engelmann spruce phloem (α-pinene and 3-carene) reduced mycelial growth rates relative to nonexposed controls, and exposure to concentrations above 1% resulted in severe-to-complete growth inhibition (Fig. 3c). Although monoterpene concentrations in colonized spruce billets were not quantified recent studies report constitutive monoterpene concentrations of ~1% (m/m) in Engelmann spruce phloem (Davis et al. 2018), indicating that B. bassiana was likely exposed to at least somewhat fungistatic monoterpene concentrations during field trials. In addition, our results indicate that α-pinene is potentially more toxic to B. bassiana than 3-carene. Likewise, interactions between B. bassiana and microbial species present in the subcortical environment may limit the ability of the fungus to kill D. rufipennis. Most adult beetles are associated with fungal symbionts including Leptographium abietinum and yeast species (Six and Bentz 2003, Cardoza et al. 2008); beetle-associated yeasts have been demonstrated to suppress growth of B. bassiana (Davis et al. 2011). However, our results indicate that the isolate used in the present study was an equal competitor to L. abietinum. Although fungal isolate of each species performed comparably in resource-capture tests, L. abietinum and/or yeasts need only maintain a small area of enemy-free space around developing larvae or pupae in order to exclude B. bassiana and limit infectivity. Another study which recently evaluated the virulence of B. bassiana toward Dendroctonus spp. tested susceptibility of red turpentine beetle [Dendroctonus valens LeConte (Coleoptera: Scolytinae)] from exotic, invasive populations in China to local isolates of the fungus (Zhang et al. 2011). The MST (4.6 d) reported from the most virulent isolate in that study was comparable to the MST reported for the isolate tested here (3–4 d depending on bioassay). However, the present study appears to be among the first to evaluate response of a Dendroctonus spp. to logs treated with conidia. One critical difference between the two studies is that MST was derived from different life stages of target insects; Zhang et al. (2011) determined mortality rates from larvae, whereas in the present study mortality rate is determined from adults, which may be generally less susceptible to B. bassiana (Hunt et al. 1984). In addition, our results indicate that responses of larvae to inoculation with B. bassiana in ex vivo studies are generally needed, where inoculant must contend with both exposure to tree or plant secondary compounds and competition with environmental, opportunistic, or even symbiotic microorganisms under a range of temperature conditions. The findings reported here and those of other studies (e.g., Behle 2006) indicate a general lack of agreement between laboratory and field studies aimed at the application of B. bassiana conidia as a biopesticide. Often, isolates which demonstrate pathogenicity in laboratory studies fail to achieve desired levels of population control in field settings. Our results suggest that one potential explanation for this disparity may be due to a mismatch between B. bassiana traits and field conditions. Accordingly, we suggest that future efforts aimed at field application of B. bassiana for management of D. rufipennis or other species could benefit from matching fungal environmental tolerance phenotypes to expected field conditions, rather than focusing solely on virulence to target insects as the criteria for isolate selection. To improve the robustness of future efforts aimed at application of B. bassiana for management of D. rufipennis in windthrown or felled P. engelmanni, we suggest that isolate selection should consider 1) growth and germination at low or variable temperatures and relative humidity, 2) ability to kill adults as well as larvae, and 3) tolerance of plant defensive compounds. Collectively, addressing these deficiencies in isolate selection may help practitioners optimize fungal isolates for both persistence under field conditions and pathogenicity, and aid in the development of new pest control products. Acknowledgments We thank the staff of the Blackrock Ranger District for assistance in selecting field sites and carrying out field studies. We are also indebted to Nathaniel Foote for assistance in the field. Funding for this work was provided by several provinces in Canada under a SERG grant and USDA Cooperative Agreement 16-CA-11046000-616. Finally, we thank the anonymous referees who reviewed this manuscript. References Cited Batta , Y. A . 2007 . Biocontrol of almond bark beetle (Scolytus amygdali Geurin-Meneville, Coleoptera: Scolytidae) using Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) . J. Appl. Microbiol . 103 : 1406 – 1414 . Google Scholar CrossRef Search ADS PubMed Behle , R. W . 2006 . Importance of direct spray and spray residue contact for infection of Trichoplusia ni larvae by field applications of Beauveria bassiana . J Econ Entomol 99 : 1120 – 1128 . Google Scholar CrossRef Search ADS PubMed Bentz , B. J. , J. Regniere , C. J. Fettig , E. M. 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Laboratory and Field Evaluation of the Entomopathogenic Fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes) for Population Management of Spruce Beetle, Dendroctonus rufipennis (Coleoptera: Scolytinae), in Felled Trees and Factors Limiting Pathogen Success

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Oxford University Press
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Published by Oxford University Press on behalf of Entomological Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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Abstract

Abstract An isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) was tested for its ability to reduce survival and reproduction of spruce beetle, Dendroctonus rufipennis (Kirby) (Coleoptera: Scolytinae), under laboratory and field conditions. Conidial suspension applied directly to adults or to filter papers that adults contacted had a median survival time of 3–4 d in laboratory assays and beetles died more rapidly when exposed to conidial suspension than when treated with surfactant solution only. In the field, conidial suspension was applied to the surface of felled and pheromone-baited Engelmann spruce (Picea engelmannii) trees using a backpack sprayer. Mortality of colonizing parent beetles (F0), reproduction (abundance of F1 offspring in logs), and emergence of F1 beetles from logs was compared between treated and nontreated logs. Application of spore suspension increased mortality of F0 adults by 36% on average. Total F1 reproduction was reduced by 17% and emergence from logs was reduced by 13% in treated logs, but considerable variability in reproduction and emergence was observed. Viable spores were re-isolated from treated logs up to 90 d after application, indicating that spores are capable of long-term persistence on the tree bole microhabitat. Subsequent in vitro tests revealed that temperatures below 15°C and exposure to spruce monoterpenes likely limit performance of B. bassiana under field conditions, but exposure to low-intensity light or interactions with spruce beetle symbiotic fungi were not strongly inhibitory. It is concluded that matching environmental tolerances of biocontrol fungi to field conditions can likely improve their usefulness for control of spruce beetle in windthrown trees. bark beetle, Beauvaria, Curculionidae, forest entomology, microbial control In the western United States, the spruce beetle, Dendroctonus rufipennis (Kirby) (Coleoptera: Scolytinae), is associated with death or decline of at least 14M Engelmann spruce (Picea engelmanii Parry ex Engelm.) trees since 1995 (Holsten et al. 1999, Wittwer 2000, Jenkins et al. 2014, Harris 2016). Endemic spruce beetle populations infest primarily weaken or stressed hosts and often kill host trees over several years through multiple re-infestations (Massey and Wygant 1954); however, high-density spruce beetle populations (‘epidemics’) preferentially attack healthy, mature spruce trees and can cause mortality within a single growing season. The transition from endemic to epidemic population phases can occur when large quantities of ‘windthrown’ trees become available to endemic populations due to blowdown events (Dyer and Taylor 1971, Schmid 1981, Raffa et al. 2008, Safranyik 2011). Windthrow occurring during winter or spring may remain green throughout the subsequent growing season and can be colonized by D. rufipennis. Colonization of windthrown spruce by beetles facilitates rapid population buildup as host material lacks the ability to mount an induced defensive response in reaction to infestation; some reports indicate that brood production of D. rufipennis in windthrown trees can exceed that in live hosts by 200–300% (Hebertson and Jenkins 2007), which may facilitate population growth beyond endemic thresholds. Consequently, methods are needed for controlling beetle population buildup in green, undefended windthrown trees. Although various approaches to beetle population control including silvicultural treatments (Sartwell and Stevens 1975, Fettig et al. 2007), chemical suppression techniques (Fettig et al. 2006), and integrated pest management tactics can reduce probability of infestation (Hansen et al. 2017), comparatively few biological methods are available. However, ‘biopesticide’ plant protectants including microorganisms are easier to license than traditional chemical means and less environmentally hazardous than commonly recommended chemical applications for beetle control such as carbaryl and pyrethroids (e.g., Bridges 2000, Ribera et al. 2001, Rohr et al. 2003). In addition, some biological alternatives to chemical application have a higher degree of host specificity and relatively shorter environmental half-life (Strasser et al. 2000, Ownley et al. 2004, Zimmerman 2007). These potential advantages have led to renewed interest in the application of biological control agents, including entomopathogenic fungi, for population control of beetles. Here, an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.-Criv.) Vuill. (1912) (Deuteromycotina: Hyphomycetes) is tested for its ability to reduce survival and population growth of D. rufipennis in P. engelmannii in the central Rocky Mountains. The use of B. bassiana and other entomopathogenic fungi for population management for pest insects has been an active area of research for decades (Feng et al. 1994, Hajek and St. Leger 1994, Meyling and Eilenberg 2007). Beauveria bassiana is an effective biological control agent against a variety of insect pests, including agricultural and stored products pests (e.g., Butt et al. 1994, Moino et al. 1998, Padin et al. 2002, Hirsch and Reineke 2014, Rondot and Reineke 2017), and B. bassiana has been tested extensively against some angiosperm-infesting bark beetles (e.g., Doberski and Tribe 1980, Doberski 1981) and other curculionids (Kreutz et al. 2004a, b; Batta 2007). Collectively, these reports indicate that certain isolates or formulations of B. bassiana may be generally effective as a biological control against wood-boring beetles. However, relatively few studies have tested the potential for B. bassiana to control populations of conifer-infesting Dendroctonus bark beetles in the field (Pabst and Sikorowski 1980, Hunt et al. 1984, Sevim et al. 2009, Zhang et al. 2011), though the fungus occurs frequently in Dendroctonus populations and habitats (Moore 1971, Ormond et al. 2010, Yao et al. 2012). Augmentation of natural entomopathogen populations may be a useful method for reducing spruce beetle reproduction in windthrown trees. Accordingly, our objectives were 1) to evaluate pathogenicity for a selected isolate in laboratory bioassays, 2) test the isolate under field conditions as a spray formulation for protection of spruce logs challenged with pheromone baits, and 3) assess environmental factors that may limit the success of field applications. Materials and Methods Isolation and Identification of Test Organisms Isolates were collected from serial dilutions of surface washes of bark, phloem, or duff taken from ponderosa (Pinus ponderosa Douglas ex C. Lawson), lodgepole pine (Pinus contorta Douglas), and Engelmann spruce; surface washes were also made from Dendroctonus ponderosae Hopkins (Coleoptera: Scolytinae) and D. rufipennis adults. Up to 10 g of material for serial dilution was surface washed in 100 ml of 0.01% Silwet L77 (Helena Chemical Company, Collierville, TN) on a table shaker for 15 min at 100 r.p.m. Serial dilutions of surface washes were spread onto potato dextrose agar amended with 0.05% dodine and 0.003% gentamicin (modified from Rangel et al. 2010) and incubated at 25°C in the dark for up to 15 d. For colonies sporulating on dodine agar and with colony morphology consistent with descriptions for Beuavaria bassiana, single colony forming units were inoculated onto Sabouraud Dextrose Agar (SDA) and allowed to sporulate for entomopathogenecity tests. For select isolates, entomopathogenecity was confirmed in a preliminary laboratory bioassay. D. rufipennis adults were captured using Lindgren funnel traps baited with pheromone lures containing frontalin, 1-methylcyclohex-2-en-1-ol (MCOL), and a proprietary blend of host tree volatiles (monoterpenes; Synergy Semiochemicals, Victoria, British Columbia, Canada) and placed individually into 6 cm diameter Petri dishes. A sterile 10-µl inoculating loop was wetted in 0.01% Silwet L77 and pressed into sporulated colonies to collect spores; spores were transferred to beetles by contact with the inoculating loop and beetles were checked daily for evidence of sporulation during a 7-d period. If sporulation was noted, single colonies were re-isolated onto potato dodine agar and transferred to SDA and again allowed to sporulate. Conidial spores were harvested into cryovials containing 30% glycerol and stored at −30°C until used. In total, 30 pure cultures obtained in this way were selected for genotyping of the 28S gene (318 bp) at a commercial facility (MIDI Labs, Newark, DE), and all were consistent with B. Bassiana sensu lato (>99% sequence similarity to B. bassiana M9344; GenBank AB576868)—since recent studies indicate cryptic speciation in the Beauvaria genus (Rehner et al. 2011), we do not assign a molecular characterization beyond the broad sense. A single putative B. bassiana isolate was selected for further laboratory and field testing on the basis of abundant conidia production and observed pathogenicity to D. rufipennis. Spore Production for Bioassays Spore preparations for laboratory and field testing were produced using solid substrate culture. Culture substrate was dehulled barley wetted with a salt solution (MgSO4: 0.5 g/liter, NaCl: 0.1 g/liter, KCl: 0.1 g/liter, FeCl3: 0.02 g/liter, (NH4)2HPO4: 5g/liter, and yeast extract 1.0 g/liter) to a final moisture content of 55% (substrate is moist, but there is no freestanding water). Salt solution was amended to the substrate and periodically mixed for 1 h to ensure complete absorption of salts into barley substrate. The mixture was autoclaved and the selected isolate was inoculated into the mixture by aseptic transfer of yeast extract broth containing the selected B. bassiana isolate at a ratio of 10 ml inoculum broth/100 g substrate. Solid cultures of ~500 g were incubated in bags aerated with sterile air at a constant flow of 500 ml/min. Sporulation and subsequent spore harvest occurred 7–10 d following aeration. To harvest spores, cultures were transferred to perforated aluminum trays and placed in a chamber with a constant flow of dehumidified, filtered air. Cultures dried within 48 h and were processed for spore recovery by sieving through 250-micron mesh. Spore concentration in dry cultures ranged from 5 × 109 to 4 × 1010 spores/g. Dry culture was milled to produce spore powder. Conidial viability from milled and sieved culture was determined by germination test. The ratio of germinated to ungerminated conidia was determined by microscope count of conidia incubated on water agar for 16 h. Conidia were considered germinated if a visible germ tube with length greater than the conidia diameter was observable, and approximately 200 conidia were counted from aggregate sieved culture (Milner et al. 1991). The viability of conidia prepared from solid substrate culture was determined to be >90%. Conidia powder diluted into a surfactant solution was used for laboratory bioassays. At a concentration of 1 mg conidia powder/1 ml 0.01% Silwet L77, the concentration of colony forming units (CFUs) was 2.4 × 108 CFU/ml. For field trials, conidia powder was mixed 1:5 in a clay-based carrier and the formulation was diluted into water with 0.01% Silwet L77 to a final concentration of 1.5 × 109 CFU/ml for spray treatment. Pathogenicity of B. bassiana to D. rufipennis in Laboratory Bioassays Using conidia produced from solid substrate culture, two laboratory assays were performed to evaluate the efficacy of the selected B. bassiana isolate for causing mortality in D. rufipennis: 1) application of conidial suspension, applied to individual beetles, and 2) application of conidial suspension to filter papers on which beetles were confined for the duration of the observation period. Both tests took place in the dark at an ambient temperature of 23°C and relative humidity ~30%. In the first test, forty new adult beetles were excavated from infested P. engelmannii material and placed individually into 3-cm diameter Petri dishes. A B. bassiana conidial suspension (1.8 × 109 CFU/ml) was directly applied to the ventral surface of the thorax and abdomen of 20 beetles in 20-µl aliquots using a pipette (treatment group), and the remaining 20 beetles were treated with 20-µl aliquots of sterile distilled water as a control. Beetles were checked daily for mortality, and the number of days until death was recorded for each individual. In the second test, each replicate consisted of 10 test beetles (as above, freshly excavated from infested P. engelmannii material) placed in 6-cm diameter Petri dishes containing filter papers (Whatman Grade 6, 4.25-cm diameter). For half the replicates (n = 10), 1 ml of B. bassiana conidial suspension (1.8 × 109 CFU/ml) was applied to the filter paper (treatment group); for the remaining replicates (n = 10), 1 ml of sterile distilled water was applied to filter papers as a control. As in the first test, cumulative beetle mortality within each Petri dish was recorded until all beetles had died. Field test of B. bassiana formulation on D. rufipennis performance The field test took place in northern Wyoming in the vicinity of Togwotee Pass (2,945 m a.s.l., approximate location: 43.825704°N, 110.214377°W). In the study area, spruce beetle population pressure was significant, with ongoing high-density D. rufipennis populations causing extensive Engelmann spruce mortality. Stands of Engelmann spruce were selected for the field test based on recent observations of tree mortality from D. rufipennis activity. Post experiment spruce beetle risk rating ranged from low-moderate to moderate based on the criteria of Schmid and Frye (1977). In total, 36 individual spruce trees were tagged for study and selected for felling; mean tree diameter at breast height of study trees was 36 ± 0.4 cm SE. During June 2016, trees were felled at 0.5 m from the ground and baited with spruce beetle pheromone lure containing frontalin, MCOL, and a proprietary blend of host tree volatiles (monoterpenes; Synergy Semiochemicals). Baits were placed in a shaded portion of the center of the bole of felled trees. Conidial suspension treatment was applied to half (n = 18) of the felled trees, and the remaining trees (n = 18) were left as nontreated controls. Conidial suspension was applied to downed trees using a Solo model 433 motorized backpack equipped with a ‘dual Y’ fan nozzle. Total application volume ranged from approximately 4–5 liters per tree, and the suspension was applied to all visible tree surfaces, with careful attention to application where tree boles contacted the ground; the ground surface near each treatment tree was treated as well. In September 2016, all study trees were cut into three 40-cm logs starting from the base; remaining tree material (upper bole and crown) was bucked and piled. Bark samples were collected from six treated trees using a 5-cm hole saw; samples were surface washed and serially diluted to estimate CFUs B. bassiana/cm2 bark. One of the three logs were randomly chosen for processing in the field to estimate mortality of colonizing F0 beetles, the remaining two logs were sealed at the ends with paraffin wax and returned to the laboratory for rearing. For logs chosen to quantify F0 mortality, three 40 × 8 cm strips (320 cm2) were cut from the bark using a bark grinder. All live and dead D. rufipennis adults and all larvae were counted within each strip and counts were normalized to express percent mortality. Strips counted in this way were treated as subsamples, and percent mortality values from strips were averaged for each log. Logs returned to the laboratory were used to determine both survival and emergence of F1 beetles. Logs were placed in rearing containers ventilated with 1 × 1-mm mesh and kept at 23°C. Logs accumulated ~800 degree-days (base 10°C) in the laboratory, after which point half the logs from each study tree (n = 18 treatment logs, and n = 18 control logs) were randomly selected for collection of F1 adults. Bark was removed from logs selected for collection of F1 adults using a draw knife, and all adult D. rufipennis were collected and counted. Beetle abundances were standardized to the surface area (beetles/dm2) of each log. Ips pilifrons (Swaine) (Coleoptera: Curculionidae) beetles also emerged from logs in notable quantities, so were collected and quantified as well . The remaining logs (n = 18 treatment logs, and n = 18 control logs) were placed outside at Waverly Experimental Ranch in northern Colorado (Colorado State University) for winterization from November 2016 to February 2017. Logs were kept in rearing containers during winterization. After 95 d (February 2017), rearing containers containing logs were returned to the laboratory and placed at 23°C. Rearing containers were checked every 48 h for newly emerged adults. Only D. rufipennis and I. pilifrons, which emerged in significant abundances from logs and were also considered in statistical analyses, were collected and counted, and counts were standardized to the surface area (beetles/dm2) of each log. Collection of emerging F1 beetles continued until no further emergence was observed. Factors Limiting Growth of B. bassiana The B. bassiana isolate used in laboratory and field entomopathogenicity tests was also subjected to a panel of in vitro assays to address isolate responses to environmental and biotic factors hypothesized to limit efficacy in the field. Specifically, the response of the isolate to 1) variation in temperature, 2) variable photoperiods, 3) exposure to secondary chemicals in the spruce subcortical environment (monoterpenes), and 4) interactions with the spruce beetle symbiotic fungus Leptographium abietinum were tested. 1) Temperature. Three replicates of the B. bassiana isolate were grown on Petri dishes containing 20% MEA in dark incubation chambers maintained at 5, 10, 15, 20, 23, 25, 30, and 35°C. Radial growth of replicates was recorded daily for 15 d or until hyphal growth reached the end of dishes. 2) Photoperiod. Five replicates were of the B. bassiana isolate were grown on petri dishes containing 20% MEA at 23°C (the optimal temperature identified for growth) in incubation chambers maintained at four photoperiods; 0, 6, 12, or 24 h at a constant light intensity of 14 µmol/m2/s (Sylvania Supersaver Cool White F40CW/SS 34Watt). This is a low light intensity that is about 5% of that experienced in full sunlight. Radial growth of replicates was recorded daily for 15 d or until hyphal growth reached the end of dishes. 3) Monoterpenes. Five replicates of the B. bassiana isolate were grown in 20% MEA at 23°C amended with one of two monoterpenes found in the phloem of all Engelmann spruce tested thus far, (+)-α-pinene (98% purity, Sigma-Aldrich) and (+)-3-carene (>90% purity, Sigma-Aldrich), at three concentrations including 0.1, 1, and 5% (v/v), consistent with constitutive (0.1–1%) and induced (5%) monoterpene concentrations (Davis et al. 2018). Radial growth of replicates was recorded daily for 15 d or until hyphal growth reached the end of dishes. 4) Interactions with L. abietinum. A final test considered the ability of B. bassiana to compete with the spruce beetle symbiotic fungus Leptographium abietinum (Peck) M.J. Wingf.; the fungus is associated with nearly all spruce beetles tested (>95%) from across the range (Six and Bentz 2003) and may be able to prevent pathogenic or opportunistic fungi from occupying the subcortical gallery environment. To assess the potential for each fungus to capture and maintain resource area when co-occurring, 10 replicates of the B. bassiana isolate were competed against 10 unique isolates of L. abietinum (isolation protocol described in Davis et al. 2018) by inoculating each fungus equidistant onto Petri dishes containing 20% MEA, and trials occurred at 23°C in the dark. Isolates were allowed to grow for 20 d after which point they were scored by scanning dishes (Epson V600) and analyzing images using the software ImageJ (Schneider et al. 2012) to estimate the proportion (%) of each dish occupied by each fungus. Data Analysis In both laboratory assays, time-until-death following treatment (median survival time, MST) was analyzed using the Kaplan–Meier estimator (Kaplan and Meier 1958) to test for differences in the survival function due to treatment effects using a log-rank test approximated by a chi-square distribution and implemented in the R add-on package ‘survival’ (Therneau and Lumley 2017). In field trials, the response variables of F0 percent mortality, F1 survival in logs, and F1 emergence from logs were analyzed using a two-sample Student’s t-test to compare trees treated with spore suspension to nontreated control trees, with each log treated as an experimental replicate. In in vitro assays testing B. bassiana responses to varying environmental conditions, the response of radial growth rate (mm/d) was analyzed using one-way ANOVA to test the fixed effects of temperature (5, 10, 15, 20, 23, 25, 28, 30, and 35°C), photoperiod (0, 6, 12, and 24 h), and concentration (0.1, 1, and 5%) of monoterpenes. A two-sample Student’s t-test was used to compare the mean proportion of total Petri dish area occupied by B. bassiana and L. abietinum in resource competition tests. Data were checked for adherence to assumptions of normality and equal variances, and all response variables conformed to these assumptions. All parametric statistical tests were performed using the software JMP 12.0 (SAS Institute, Cary, NC) and a type I error rate of α = 0.05. Results Pathogenicity of B. bassiana to D. rufipennis in Laboratory Bioassays Direct application of spore suspension to D. rufipennis adults in the laboratory significantly impacted MST of beetles, and beetles treated with spore suspension died 20% more rapidly on average (MST: 4 d) than beetles treated only with distilled water (MST: 5 d; χ2= 4.113, df = 1, n = 40, P = 0.042; Fig. 1a). Similarly, application of spore suspension to filter papers contacted by D. rufipennis adults significantly reduced beetle survival time by 33% on average, with an MST of 43.0 d for beetles exposed to filter papers treated with spore suspension compared with an MST of 6 d for beetles exposed to filter papers treated with only water (χ2 = 72.878, df = 1, n = 200, P < 0.001; Fig. 1b). Fig. 1. View largeDownload slide Kaplan–Meier survivorship curves reflecting time-until-death for D. rufipennis in laboratory assays following a) direct application of spore suspension to beetles, or b) application of spore suspension to filter papers contacted by adult beetles. Solids lines and closed circles denote B. bassiana treatments, dashed lines, and open circles denote control treatments; bars show plus or minus on standard error of the mean. Fig. 1. View largeDownload slide Kaplan–Meier survivorship curves reflecting time-until-death for D. rufipennis in laboratory assays following a) direct application of spore suspension to beetles, or b) application of spore suspension to filter papers contacted by adult beetles. Solids lines and closed circles denote B. bassiana treatments, dashed lines, and open circles denote control treatments; bars show plus or minus on standard error of the mean. Field Test of B. bassiana Formulation on D. rufipennis Performance In total, 1,404 living and 978 dead D. rufipennis adults were counted from removed bark strips. Treatment with clay-based spore suspension reduced survival of adult D. rufipennis colonizing and ovipositing in baited host material by 36% on average, and this difference was statistically significant (t34 = 4.324; P < 0.0001; Fig. 2a). However, no evidence was found for reduced survival of larvae (t34 = 0.227; P < 0.821) resulting from application of spore suspension, and it was not possible to separate Ips from Dendroctonus larvae in field counts; thus, evaluation of spore suspension on larval survival in the field considers the sum of Dendroctonus and I. pilifrons larvae. Fig. 2. View largeDownload slide a) Relative mortality of colonizing adult (F0) D. rufipennis following entry into logs treated with a clay-based spore suspension of B. bassiana (treatment group) or nontreated control logs. b) Brood (abundance of F1 offspring) counted in logs, and (c) emergence of brood from logs. Open gray circles denote sample data, closed black circles denote sample means, and bars show one standard error. Asterisk denotes significant (P < 0.05) differences between treatment and control. Fig. 2. View largeDownload slide a) Relative mortality of colonizing adult (F0) D. rufipennis following entry into logs treated with a clay-based spore suspension of B. bassiana (treatment group) or nontreated control logs. b) Brood (abundance of F1 offspring) counted in logs, and (c) emergence of brood from logs. Open gray circles denote sample data, closed black circles denote sample means, and bars show one standard error. Asterisk denotes significant (P < 0.05) differences between treatment and control. In total, 3,989 D. rufipennis and 1,557 I. pilifrons brood were produced in logs peeled for quantification of reproduction (i.e., abundance of F1 offspring in logs prior to emergence). Mean D. rufipennis brood (F1) abundance in spruce logs was 3.4 beetles/dm2 in nontreated logs, and 2.7 beetles/dm2 in logs treated with spore suspension, but this difference was not statistically significant (t31 = −0.885, P = 0.382; Fig. 2b). Similarly, reproduction of I. pilifrons in spruce logs was not significantly affected by treatment with spores (t26 = 0.396, P = 0.695), and I. pilifrons abundance in nontreated logs was 1.3 beetles/dm2, whereas abundance in treated logs was 1.7 beetles/dm2. The sum of D. rufipennis adults and I. pilifrons adults was also not significantly affected by treatment of logs with spores (t26 = 0.075, P = 0.785). Mean emergence of D. rufipennis F1 brood from winterized spruce logs was nearly two orders of magnitude less than what was counted from peeled logs during evaluation of reproduction, with only 208 adult D. rufipennis emerging (~5% emergence). Although there was a 13% reduction in D. rufipennis emergence from treated versus nontreated logs, this result was not statistically significant (t35 = 0.017, P = 0.893; Fig. 2c), and no D. rufipennis brood emerged from 44% of logs (16 of 36 experimental units). Similarly, emergence of I. pilifrons (t35 = 0.233, P = 0.632) and the sum of D. rufipennis and I. pilifrons (t35 = −0.492, P = 0.625) were not affected by treatment of logs with spore suspension. Analysis of bark samples from treated logs indicated that immediately following initial treatment viable CFUs ranged from 5.3 × 106–2.3 × 107 viable CFU/cm2 bark (mean = 1.0 × 107); however, after 90 d in the field, viable CFUs/cm2 bark averaged 1.5 × 105. Factors Limiting Growth of B. bassiana 1) Temperature. Temperature had a considerable effect on variation in mean B. bassiana radial growth rates (F7, 16 = 15.528; P < 0.001). Growth rate was maximized at 23°C in the dark but was comparable across temperatures ranging from 20–30°C. There was a significant decline in growth rate at 15°C, and growth at 10°C was reduced by84% on average in comparison with growth at 15°C and by 94% on average in comparison with growth at 23°C. Mycelia failed to establish at 5 and 35°C, indicating the lower and upper bounds of growth for the isolate (Fig. 3a). 2) Photoperiod. There was no effect of photoperiod (0, 6, 12, or 24 h) on mycelial growth of B. bassiana at the light intensity tested (F3, 14 = 2.177; P = 0.013; Fig. 3b). 3) Monoterpenes. Inoculation into dishes amended with either monoterpene uniformly retarded B. bassiana growth (α-pinene test: F2, 12 = 331.790; P < 0.001; 3-carene test: F2, 12 = 7.657; P = 0.007); at constitutive concentrations (0.1–1%) the test isolate was still capable of growing at 70–80% of the rate of mycelia growing in the absence of monoterpenes. However, at concentrations that might be expected in defensively induced Engelmann spruce trees (5%), growth in media amended with α-pinene or 3-carene was completely suppressed or reduced by 83% on average, respectively (Fig. 3c). 4) Interactions with L. abietinum. The test isolate of B. bassiana appeared to be an equal competitor with L. abietinum in a resource limited environment, with neither species occupying a greater proportion of resource area on average (t9 = 0.249; P = 0.808; Fig. 3d). Fig. 3. View largeDownload slide Effects of multiple environmental conditions on the in vitro growth performance of B. bassiana test isolate, including a) temperature variability; b) photoperiod; c) exposure to monoterpenes; and d) interactions with the spruce beetle symbiotic fungus, L. abietinum. Lettering denotes Tukey’s HSD test, and bars show plus or minus one standard error of the mean. Aside from data summarized in (a), all tests were performed at 23°C. Fig. 3. View largeDownload slide Effects of multiple environmental conditions on the in vitro growth performance of B. bassiana test isolate, including a) temperature variability; b) photoperiod; c) exposure to monoterpenes; and d) interactions with the spruce beetle symbiotic fungus, L. abietinum. Lettering denotes Tukey’s HSD test, and bars show plus or minus one standard error of the mean. Aside from data summarized in (a), all tests were performed at 23°C. Discussion Laboratory tests indicated that the selected B. bassiana isolate was entomopathogenic; treatment with conidial suspension caused a decrease in median adult D. rufipennuis survival time of 21–40%, depending on the bioassay (Fig. 1a and b). The effect of treatment on reducing D. rufipennis MST was greater in the assay where beetles merely contacted treated surfaces (filter papers), which is the same mode of application used in treating downed trees. Although the tested B. bassiana isolate was effective at killing D. rufipennis adults contacting treated surfaces under laboratory conditions, this did not translate to field efficacy. In field tests, mortality of parent beetles (F0) colonizing highly attractive (pheromone-baited) felled trees after contact with surface treatments was significantly higher than in nontreated felled trees (Fig. 2a); however, this effect did not result in a reduction to F1 brood size (Fig. 2b) or emergence of brood from billets (Fig. 2c). Various environmental effects may limit the efficacy of B. bassiana in pest control applications and could potentially explain the disparity observed here between laboratory and field tests. For instance, ambient temperature and relative humidity interact to impact infection rates in larvae of the elm bark beetle, Scolytus scolytus (F.) (Coleoptera: Scolytinae) by B. bassiana (Doberski 1981), with fungal performance generally optimized at temperatures between 20 and 25°C. Exposure to ultraviolet light (particularly UV-B radiation) is also considered detrimental to the growth and persistence of the fungus (Inglis et al. 1995). These two factors are often reported as primary limitations to B. bassiana efficacy in the field. In the present study, environmental conditions over the duration of the field trial did not match optimal conditions determined by a posteriori laboratory assays. Daily temperature and humidity records summarized at a 4-km spatial resolution from June–August (2016), the temporal window during which experimental treatments and D. rufipennis flight and host colonization occurred in our study area, showed that mean temperature and relative humidity during field tests were approximately 13.5°C and 37%, respectively (PRISM Climate Group 2018). However, our in vitro experiments indicate that the optimal thermal range for the tested B. Bassiana isolate were between 20–30°C, with an 84% decline in mycelial growth when ambient temperatures decreased from 15 to 10°C (Fig. 3a). Exposure to all tested low-intensity photoperiods (6–24 h at 14 µmol/m2 min) had a weak-to-modest negative effect (9–17% reduction) on mycelial growth on average, but this was not statistically significant. In addition to suboptimal environmental conditions, Dendroctonus spp. may be low-quality hosts for B. bassiana. Hunt et al. (1984) determined that germination of conidia is suppressed on the cuticle of mountain pine beetle (D. ponderosae) due to nutrient deficiencies. The sclerotized cuticle lacks essential nutrients required for B. bassiana germination, although beetle hemolymph is a sufficient to induce extensive germination. Accordingly, adult beetles lacking entry points into the hemolymph may be somewhat resistant to colonization by the fungus, particularly if spores or new germinants are immediately exposed to tree secondary chemicals. B. bassiana infecting Dendroctonus spp. must contend with exposure to tree secondary compounds, especially, monoterpenes, which may permeate the subcortical egg-gallery environment and can inhibit growth of many fungal species (Hofstetter et al. 2005, Davis and Hofstetter 2012, Marei et al. 2012). Several studies have demonstrated that exposure to fungistatic compounds on the phylloplane can reduce efficacy of entomopathogenic fungi (e.g., Inyang et al. 1998, Poprawski and Jones 2001). Exposure to all tested concentrations (0.1, 1, and 5% v/v) of two monoterpenes commonly found in Engelmann spruce phloem (α-pinene and 3-carene) reduced mycelial growth rates relative to nonexposed controls, and exposure to concentrations above 1% resulted in severe-to-complete growth inhibition (Fig. 3c). Although monoterpene concentrations in colonized spruce billets were not quantified recent studies report constitutive monoterpene concentrations of ~1% (m/m) in Engelmann spruce phloem (Davis et al. 2018), indicating that B. bassiana was likely exposed to at least somewhat fungistatic monoterpene concentrations during field trials. In addition, our results indicate that α-pinene is potentially more toxic to B. bassiana than 3-carene. Likewise, interactions between B. bassiana and microbial species present in the subcortical environment may limit the ability of the fungus to kill D. rufipennis. Most adult beetles are associated with fungal symbionts including Leptographium abietinum and yeast species (Six and Bentz 2003, Cardoza et al. 2008); beetle-associated yeasts have been demonstrated to suppress growth of B. bassiana (Davis et al. 2011). However, our results indicate that the isolate used in the present study was an equal competitor to L. abietinum. Although fungal isolate of each species performed comparably in resource-capture tests, L. abietinum and/or yeasts need only maintain a small area of enemy-free space around developing larvae or pupae in order to exclude B. bassiana and limit infectivity. Another study which recently evaluated the virulence of B. bassiana toward Dendroctonus spp. tested susceptibility of red turpentine beetle [Dendroctonus valens LeConte (Coleoptera: Scolytinae)] from exotic, invasive populations in China to local isolates of the fungus (Zhang et al. 2011). The MST (4.6 d) reported from the most virulent isolate in that study was comparable to the MST reported for the isolate tested here (3–4 d depending on bioassay). However, the present study appears to be among the first to evaluate response of a Dendroctonus spp. to logs treated with conidia. One critical difference between the two studies is that MST was derived from different life stages of target insects; Zhang et al. (2011) determined mortality rates from larvae, whereas in the present study mortality rate is determined from adults, which may be generally less susceptible to B. bassiana (Hunt et al. 1984). In addition, our results indicate that responses of larvae to inoculation with B. bassiana in ex vivo studies are generally needed, where inoculant must contend with both exposure to tree or plant secondary compounds and competition with environmental, opportunistic, or even symbiotic microorganisms under a range of temperature conditions. The findings reported here and those of other studies (e.g., Behle 2006) indicate a general lack of agreement between laboratory and field studies aimed at the application of B. bassiana conidia as a biopesticide. Often, isolates which demonstrate pathogenicity in laboratory studies fail to achieve desired levels of population control in field settings. Our results suggest that one potential explanation for this disparity may be due to a mismatch between B. bassiana traits and field conditions. Accordingly, we suggest that future efforts aimed at field application of B. bassiana for management of D. rufipennis or other species could benefit from matching fungal environmental tolerance phenotypes to expected field conditions, rather than focusing solely on virulence to target insects as the criteria for isolate selection. To improve the robustness of future efforts aimed at application of B. bassiana for management of D. rufipennis in windthrown or felled P. engelmanni, we suggest that isolate selection should consider 1) growth and germination at low or variable temperatures and relative humidity, 2) ability to kill adults as well as larvae, and 3) tolerance of plant defensive compounds. Collectively, addressing these deficiencies in isolate selection may help practitioners optimize fungal isolates for both persistence under field conditions and pathogenicity, and aid in the development of new pest control products. Acknowledgments We thank the staff of the Blackrock Ranger District for assistance in selecting field sites and carrying out field studies. We are also indebted to Nathaniel Foote for assistance in the field. Funding for this work was provided by several provinces in Canada under a SERG grant and USDA Cooperative Agreement 16-CA-11046000-616. Finally, we thank the anonymous referees who reviewed this manuscript. References Cited Batta , Y. A . 2007 . Biocontrol of almond bark beetle (Scolytus amygdali Geurin-Meneville, Coleoptera: Scolytidae) using Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) . J. Appl. Microbiol . 103 : 1406 – 1414 . Google Scholar CrossRef Search ADS PubMed Behle , R. W . 2006 . Importance of direct spray and spray residue contact for infection of Trichoplusia ni larvae by field applications of Beauveria bassiana . J Econ Entomol 99 : 1120 – 1128 . Google Scholar CrossRef Search ADS PubMed Bentz , B. J. , J. Regniere , C. J. Fettig , E. M. 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Environmental EntomologyOxford University Press

Published: Mar 24, 2018

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