Select genotypes of white and green ash show heritable, elevated resistance to emerald ash borer

Two experiments were performed to determine the extent to which ash species (black, green and white) and larval developmental stage (second, third and fourth instar) affect the efficiency of phloem amino acid utilization by emerald ash borer (EAB) Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) larvae. EAB larvae generally utilized green ash amino acids more efficiently than those of the other two species. For example, the concentrations of only six (two essential) and seven (two essential) amino acids were lower in frass from EAB that fed upon black and white ash than in the corresponding phloem, respectively. By contrast, concentrations of 16 (eight essential) amino acids were lower in the frass from EAB that fed upon green ash than in the phloem. In addition, in green ash, the frass : phloem ratios of 13 amino acids were lower than their counterparts in black and white ash. The concentrations of non‐essential amino acids glycine and hydroxylproline were greater in frass than in phloem when EAB fed on black ash, although not when EAB fed on green or white ash. The concentration of total phenolics (a group of putative defensive compounds to EAB, expressed as antioxidant activity of acetone extraction) was high in EAB frass but even higher in the phloem samples when the data were pooled across ash species and EAB larval stages. This suggests EAB larvae may eliminate phenolics through a combination of direct excretion and enzymatic conversion of phenolics to nonphenolics before excretion. Because the ratio of frass total phenolics to phloem total phenolics in white ash was lower than the ratios in black and green ash, the ability to destroy phenolics or convert them to nonphenolics was greater when EAB larvae fed on white ash. Fourth‐instar EAB extracted phloem amino acids, including threonine, more efficiently than third‐instar EAB. The different larval developmental stages of EAB did not differ in their apparent ability to destroy phenolics or convert them to nonphenolics.

Herbivorous insects can have their reproductive potential influenced by the quality and species of host plants they feed upon. The emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is an invasive pest of ash trees (Fraxinus spp.) within its introduced range. As adults, EAB must feed upon foliage to sexually mature. We compared the influence of 4 North American ash species on EAB via foliage feeding to assess impacts on female lifespan and reproductive metrics. We fed 136 female EAB, 34 in each foliage group, either black, green, tropical, or white ash throughout their adult life. We performed daily inspections for adult mortality, oviposition, and egg hatching. We found that the mean female lifespan, fertility rate, and mean egg development time were not affected by the ash species. Potential and realized fecundity each increased with summed female group lifespan (days), but this rate differed among ash species. Consequently, there was a statistically significant interaction effect of the summed female group lifespan and the host foliage. Green ash-fed EAB laid (2.94 ± 0.86 eggs/female days) and hatched (1.67 ± 0.59 eggs/female days) the most eggs, more than double the rates of EAB feeding on black (1.39 ± 0.48 laid and 0.75 ± 0.30 hatched eggs/female days) and white (1.08 ± 0.35 laid and 0.65 ± 0.22 hatched eggs/female days) ash. Adults feeding on green ash resulted in the greatest EAB fecundity suggesting that the presence of green ash may promote population growth and provide a pathway to overwhelm other ash species nearby.

Nine-week-old green (Fraxinus pennsylvanica Marsh.) and white (F. americana L.) ash were exposed to O3 and/or SO2 (control, 0.10 ppm O3, 0.08 ppm SO2 or 0.10 ppm O3 + 0.08 ppm SO2) for 4 h d-1, 5 d wk-1 in combination with simulated rain (pH 3.0, 4.3 or 5.6, 1 h d-1, 2 d wk-1 at 0.75 cm h-1) for 6 weeks, under controlled laboratory conditions, with rain applied either just before or after fumigation. Across all rain treatments, white ash biomass was suppressed by the application of O3 and cumulative shoot elongation of green ash exposed to O3 and/or SO2 was less than controls. The combination of O3 + SO2 did not affect the growth of either species more than the pollutants applied alone. Leaf area ratio (LAR) and root to shoot ratio (RSR) exhibited quadratic responses to rain pH in green ash, across all pollutant treatments. Significant pollutant x pH interactions occurred in leaf weight ratio (LWR) in green ash and LAR and RSR in white ash. Significant linear increases in LAR and decreases in RSR, with decreasing pH, were observed for O3 and SO2-treated white ash. These findings are discussed relative to implications of the effects of gaseous pollutants in combination with acid rain on green and white ash growth. For. Sci. 34(4):1016-1029.

Despite catastrophic ash (Fraxinus spp.) mortality observed by the mid-2000s in the epicenter of the emerald ash borer (EAB) (Agrilus planipennis Fairmaire) invasion in southeast Michigan, we noticed numerous live white ash (Fraxinus americana L.) in some forests in this region. In 2015, we inventoried overstory trees and regeneration in 28 white ash sites spanning 11 counties. White ash survival ranged from 0% to 100% of stems. Overall, 75% of 821 white ash trees, ranging from 10.0 to 44.0 cm diameter at breast height, and 66% of the white ash basal area, ranging from 0.3 to 3.5 m2·ha–1, were alive. Nearly all live white ash had signs of previous EAB colonization, but 83% had healthy canopies (≤10% canopy dieback). Green ash trees were recorded in 27 sites, but 92% were killed by EAB. Model selection indicated that variation in white ash survival was related to white ash abundance and distance of sites from the original EAB epicenter but not to green ash related variables or to the distance of sites from the Asian parasitoid release or recovery locations. Regeneration strata were dominated by white ash, suggesting that some white ash populations may persist in post-invasion areas.

Growth of white ash (Fraxinusamericana L.), green ash (Fraxinuspennsylvanica Marsh.), and velvet ash (Fraxinusvelutina Torr.) as related to infection by ash yellows mycoplasmalike organisms (MLOs) was assessed by diagnosing and grouping trees as MLO infected or healthy and comparing their previous annual radial growth over periods of 9–22 years. White ash and green ash 17–30 years old were studied on two sites per species in central New York State. Velvet ash 30–90 years old was studied in Zion National Park, Utah. MLOs were detected by fluorescence microscopy. Widths of growth rings were measured on increment cores or cross sections of main stems. Growth trends of diseased and healthy groups of white ash and green ash diverged when ash stands were 12–21 years old. In the fifth year after divergence, the unweighted average growth of diseased green ash across two plots was 70% of that of healthy trees. For white ash, the corresponding average was 61%. In white ash tested annually for MLO infection, growth reduction preceded MLO detection by an average of 1 year. Velvet ash did not sustain MLO-associated growth reduction. This species may be tolerant of infection by ash yellows MLOs.

The Emerald Ash Borer (EAB), Agrilus planipennis, Fairmaire, an Asian invasive alien buprestid has devastated tens of millions of ash trees (Fraxinus spp.) in North America. Foliar phytochemicals of the genus Fraxinus (Oleaceae): Fraxinus pennsylvanica (Green ash), F. americana (White ash), F. profunda (Bush) Bush. (Pumpkin ash), F. quadrangulata Michx. (Blue ash), F. nigra Marsh. (Black ash) and F. mandshurica (Manchurian ash) were investigated using HPLC-MS/MS and untargeted metabolomics. HPLC-MS/MS help identified 26 compounds, including phenolics, flavonoids and coumarins in varying amounts. Hydroxycoumarins, esculetin, esculin, fraxetin, fraxin, fraxidin and scopoletin were isolated from blue, black and Manchurian ashes. High-throughput metabolomics revealed 35 metabolites, including terpenes, secoiridoids and lignans. Metabolomic profiling indicated several upregulated putative compounds from Manchurian ash, especially fraxinol, ligstroside, oleuropin, matairesinol, pinoresinol glucoside, 8-hydroxypinoresinol-4-glucoside, verbenalin, hydroxytyrosol-1-O-glucoside, totarol and ar-artemisene. Further, dicyclomine, aphidicolin, parthenolide, famciclovir, ar-turmerone and myriocin were identified upregulated in blue ash. Principal component analysis demonstrated a clear separation between Manchurian and blue ashes from black, green, white and pumpkin ashes. The presence of defensive compounds upregulated in Manchurian ash, suggests their potential role in providing constitutive resistance to EAB, and reflects its co-evolutionary history with A. planipennis, where they appear to coexist in their native habitats.

Emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is an aggressive invader from Asia that has killed millions of trees in North America, causing substantial ecosystem effects and economic losses. All North American ash, Fraxinus spp., are thought to be susceptible, but recently emerald ash borer has been documented developing in a novel host, white fringetree, Chionanthus virginicus. We evaluated larval performance in two common ash species and white fringetree by infesting bolts with emerald ash borer eggs. In addition we evaluated cambial nitrogen, carbon, carbon:nitrogen, stem density, and response to artificial wounding, to determine which host plant characteristics most influence larval development. We also conducted choice and no choice assays using the classical biological control agent, Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae), to assess its ability to locate larval emerald ash borer in the different host plants. We found significantly lower survival rates of emerald ash borer larvae in white fringetree compared to white ash, F. americana. Larval phloem consumption and larval growth were lower in fringetree than in either white or blue ash, F. quadrangulata. Carbon content and density were greater in fringetree than in either ash species. Response to wounding, measured by callus tissue formation, was greatest in white ash. In choice assays, T. planipennisi only parasitized emerald ash borer larvae in ash bolts, and in no-choice tests failed to parasitize larvae in fringetree. Our findings corroborate studies showing that fringetree is a suitable host for emerald ash borer larvae. Failure of T. planipennisi to parasitize larvae within fringetree has implications for the efficacy of this classical biological control agent in regulating emerald ash borer populations. Coupled with the use of white fringetree as a reservoir host, the enemy free space provided to emerald ash borer through use of this alternate host may have significant repercussions for emerald ash borer invasion dynamics.

Emerald ash borer (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae), a phloem-feeding insect native to Asia, was identified in 2002 as the cause of widespread ash (Fraxinus) mortality in southeast Michigan, U.S. and Windsor, Ontario, Canada. Little information about A. planipennis is available from its native range and it was not known whether this invasive pest would exhibit a preference for a particular North American ash species. We monitored A. planipennis density and canopy condition on green ash (F. pennsylvanica) and white ash (F. americana) street trees in four neighborhoods and on white and blue ash (F. quadrangulata) trees in two woodlots in southeast Michigan. Green ash street trees had significantly more canopy dieback and higher A. planipennis densities than white ash trees growing in the same neighborhood. Density increased by two- to fourfold in both species over a 3-year period. Canopy dieback increased linearly from 2002 to 2005 as A. planipennis density increased (R 2= 0.70). In each of the woodlots, A. planipennis densities were significantly higher on white ash trees than blue ash trees. Woodpecker predation occurred in all sites and accounted for 35% of the A. planipennis that developed on trees we surveyed. Results indicate that surveys for A. planipennis detection in areas with multiple ash species should focus on the relatively preferred species.

Emerald ash borer (Agrilus planipennis Fairmaire) was recently found on a novel host in North America, white fringetree (Chionanthus virginicus L.) (Oleaceae). In this study, we artificially infested 4-yr-old, naïve white fringetree and white ash (Fraxinus americana L.) saplings under well-watered and water-deficit conditions with emerald ash borer eggs. We used physiological and phenotypical approaches to investigate both plant response to emerald ash borer and insect development at 21, 36, and 61 d postinfestation. Photosynthesis was reduced in both tree species by larval feeding, but not by water deficits. Emerald ash borer larvae established and survived successfully on white ash. Both establishment and survival were lower on white fringetree than on white ash. Larvae were larger, and had reached higher instars at all three time points on white ash than on white fringetrees. Larvae grew faster in white ash under water-deficit conditions; however, water-deficit conditions negatively impacted survival of larvae at 61 d postinfestation in white fringetrees, although head size did not differ among surviving larvae. White ash showed higher callus formation in well-watered trees, but no impact on larval survival was observed. In white fringetree, callus formation was not affected by water treatment, and was inversely related to larval survival. The higher rate of mortality and slow growth rate of larvae in white fringetree as compared to white ash suggest that populations of emerald ash borer may be sustained by white fringetree, but may grow more slowly than in white ash.

The emerald ash borer (EAB; Agrilus planipennis Fairmaire; Coleoptera: Buprestidae), is an exotic wood-boring beetle that has been threatening North American ash (Fraxinus spp.) resources since its discovery in Michigan and Ontario in 2002. In this study, we investigated the phytochemical responses of the three most common North American ash species (black, green, and white ash) in northeastern USA to EAB adult feeding. Black ash was the least responsive to EAB adult feeding in terms of the induction of volatile compounds, and levels of only two (indole and benzyl cyanide) of the 11 compounds studied increased. In green ash, levels of two [(E)-β-ocimene and indole] of the 11 volatile compounds studied were elevated, while the levels of two green leaf volatiles [hexanal and (E)-2-hexenal] decreased. White ash showed the greatest response with an increase in levels of seven of the 11 compounds studied. Qualitative differences among ash species were detected. Among the phenolic compounds detected, ligustroside was the only one detected in all three species. Oleuropein aglycone and 2 unidentified compounds were found only in black ash; coumaroylquinic acid and feruloylquinic acid were detected only in green ash; and verbascoside hexoside was detected only in white ash. EAB adult feeding did not elicit or decrease concentrations of any selected individual phenolic compounds. However, although levels of total phenolics from black and green ash foliage were not affected by EAB adult feeding, they decreased significantly in white ash. EAB adult feeding elevated chymotrypsin inhibitors in black ash. The possible ecological implications of these findings are discussed.

All North American ash (Fraxinus spp.) species are threatened by the emerald ash borer (EAB; Agrilus planipennis), an exotic beetle which has already destroyed millions of ash trees in the U.S. and Canada. Although both chemical insecticides and biological control can be effective, and host resistance appears possible, the speed of the invasion has defied traditional management approaches. One potential, innovative approach to managing this destructive insect is to develop a host tree-induced gene silencing strategy using RNA interference (RNAi) constructs targeting EAB-specific genes. An important requirement for applying RNAi technology is a reliable transformation/regeneration system for the host tree species. We developed an Agrobacterium-mediated gene transfer system for white ash (F. americana) and green ash (F. pennsylvanica) using the embryogenic cultures of these species as target material. Embryogenic suspension cultures of multiple genotypes of both species were plated and inoculated with A. tumefaciens carrying the pFHI-GUSi expression vector, which carries the nptII selectable marker and intron-GUS reporter genes, followed by selection on a semi-solid medium containing geneticin. Putative transgenic events showed expression of the GUS gene at all tested developmental stages from callus to plantlets, and transgene presence in the leaves of regenerated plants was confirmed using PCR. The overall average transformation efficiency achieved was 14.5 transgenic events per gram of tissue. Transgenic somatic seedlings of two white ash and three green ash genotypes were produced and acclimated to greenhouse conditions.

Potential for persistence of blue ash in the presence of emerald ash borer in southeastern Michigan

The emerald ash borer (Agrilus planipennis Fairmaire, EAB) is an alien, invasive wood-boring insect that is responsible for killing millions of ash trees since its discovery in North America in 2002. All North American ash species (Fraxinus spp.) that EAB has encountered have shown various degrees of susceptibility, while Manchurian ash (Fraxinus mandshurica Ruprecht), which shares a co-evolutionary history with this insect, is resistant. Recent studies have looked into constitutive resistance mechanisms in Manchurian ash, concentrating on the secondary phloem, which is the feeding substrate for the insect. In addition to specialized metabolism and defense-related components, primary metabolites and nutritional summaries can also be important to understand the feeding behavior of insect herbivores. Here, we have compared the nutritional characteristics (water content, total protein, free amino acids, total soluble sugars and starch, percent carbon and nitrogen, and macro- and micronutrients) of outer bark and phloem from black, green, white and Manchurian ash to determine their relevance to resistance or susceptibility to EAB. Water content and concentrations of Al, Ba, Cu, Fe, K, Li, tryptophan and an unknown compound were found to separate black and Manchurian ash from green and white ash in a principal component analysis (PCA), confirming their phylogenetic placements into two distinct clades. The traits that distinguished Manchurian ash from black ash in the PCA were water content and concentrations of total soluble sugars, histidine, lysine, methionine, ornithine, proline, sarcosine, tyramine, tyrosol, Al, Fe, K, Na, V and an unknown compound. However, only proline, tyramine and tyrosol were significantly different, and higher, in Manchurian ash than in black ash.

Green ash (Fraxinus pennsylvanica) and white ash (F. americana) populations are currently experiencing major declines across their native ranges in North America due to infestation by the exotic insect pest emerald ash borer (Agrilus planipennis). The development of a reliable method for the long-term storage of green and white ash germplasm in the form of embryogenic cultures using cryopreservation would be a considerable aid to ash conservation efforts. We compared recovery percentages of cryopreserved green and white ash embryogenic cultures using vitrification versus slow cooling methods. Three Plant Vitrification Solution 2 (PVS2) exposure durations (40, 60, and 80 min) for vitrification and three DMSO concentrations (5%, 10%, and 15%) for slow cooling were tested for their effects on the percentage of cultures that regrew following cryostorage. Vitrification resulted in a higher overall culture recovery percentage (91%) compared to cultures that were cryostored using the slow cooling approach (39%), and a more rapid initiation of regrowth (5 days versus 2–3 weeks) resulted. Recovery from cryostorage by cultures using the slow cooling approach varied significantly (p < 0.05) between experiments and with genotype (p < 0.05). The recovery of vitrified tissue from cryostorage did not vary with genotype, species, or PVS2 exposure duration (p > 0.05). The vitrification cryopreservation protocol provides a reliable and versatile alternative to the traditional slow cooling method, strengthening our ability to preserve valuable ash germplasm for conservation and restoration.

Emerald Ash Borer (EAB), Agrilus planipennis, is a beetle that originates from East Asia. Upon invasion to North America in the early 2000s, it killed untold millions of ash trees. In European Russia, EAB was first detected in Moscow in 2003 and proved to have the potential to also kill native European ash (Fraxinus excelsior). The beetle has since spread in all geographic directions, establishing itself in eastern Ukraine by 2019 and possessing potential for further westward spread towards the EU. Apart from the approaching EAB, F. excelsior is currently threatened by the dieback disease (ADB) caused by the invasive ascomycete fungus Hymenoscyphus fraxineus. The infestation by EAB combined with ADB infection is expected to be more lethal than either of them alone, yet the potential consequences are unknown. To date, eastern Ukraine represents the geographic area in which both invasions overlap, thus providing the opportunity for related investigations. The aims of the study were to investigate: (i) the EAB expansion range in Ukraine, (ii) the relative susceptibility of F. excelsior and American ash (Fraxinus pennsylvanica) to EAB and ADB, and (iii) the combined effect/impact on ash condition imposed by both the pest and disease in the area subjected to the invasion. The results have demonstrated that (i) the invasion of EAB is currently expanding both in terms of newly infested trees and invaded geographic area; (ii) F. excelsior is more resistant to EAB than F. pennsylvanica, while F. excelsior is more susceptible to ADB than F. pennsylvanica; and (iii) the infection by ADB is likely to predispose F. excelsior to the infestation by EAB. It was concluded that inventory and mapping of surviving F. excelsior, affected by both ADB and EAB, is necessary to acquire genetic resources for the work on strategic, long-term restoration of F. excelsior in devastated areas, thereby tackling a possible invasion of EAB to the EU.