Drought legacies in mixed Mediterranean forests: Analysing the effects of structural overshoot, functional traits and site factors.

Previous favorable climate conditions stimulate tree growth making some forests more vulnerable to hotter droughts. This so-called structural overshoot may contribute to forest dieback, but there is little evidence on its relative importance depending on site conditions and tree species because of limited field data. Here, we analyzed remote sensing (NDVI) and tree-ring width data to evaluate the impacts of the 2017 drought on canopy cover and growth in mixed Mediterranean forests (Fraxinus ornus, Quercus pubescens, Acer monspessulanum, Pinus pinaster) located in southern Italy. Legacy effects were assessed by calculating differences between observed and predicted basal area increment (BAI). Overall, the growth response of the study stands to the 2017 drought was contingent on site conditions and species characteristics. Most sites presented BAI and canopy cover reductions during the drought. Growth decline was followed by a quick recovery and positive legacy effects, particularly in the case of F. ornus. However, we found negative drought legacies in some species (e.g., Q. pubescens, A. monspessulanum) and sites. In those sites showing negative legacies, high growth rates prior to drought in response to previous wet winter-spring conditions may have predisposed trees to drought damage. Vice versa, the positive drought legacy found in some F. ornus site was linked to post-drought growth release due to Q. pubescens dieback and mortality. Therefore, we found evidences of structural drought overshoot, but it was restricted to specific sites and species. Our findings highlight the importance of considering site settings such as stand composition, pre-drought conditions and different tree species when studying structural overshoot. Droughts contribute to modify the composition and dynamics in mixed forests.

Genetic resources of common ash (Fraxinus excelsior L.) in Poland.

BACKGROUND: Knowledge of genetic structure and the factors that shape it has an impact on forest management practices. European ash (Fraxinus excelsior L.) has declined dramatically throughout its range as a result of a disease caused by the fungus Hymenoscyphus fraxineus. Despite the need for conservation and restoration of the species, genetic data required to guide these efforts at the country level are scarce. Thereofore, we studied the chloroplast and nuclear genetic diversity of 26 natural common ash populations (1269 trees) in Poland. RESULTS: Chloroplast polymorphisms grouped the populations into two geographically structured phylogenetic lineages ascribed to different glacial refugia (the Balkans and the Eastern Alps). However, the populations demonstrated high genetic diversity (mean AR = 12.35; mean Ho = 0.769; mean He = 0.542) but low differentiation based on nuclear microsatellites (FST = 0.045). Significant spatial genetic structure, consistent with models of isolation by distance, was detected in 14 out of 23 populations. Estimated effective population size was moderate-to-high, with a harmonic mean of 57.5 individuals per population. CONCLUSIONS: Genetic diversity was not homogeneously distributed among populations within phylogenetic gene pools, indicating that ash populations are not equal as potential sources of reproductive material. Genetic differences among populations could be related to their histories, including founder effects or gene flow between evolutionary lineages (admixture). Our results suggest that ash stands across Poland could be treated as two main management units (seed zones). Therefore, despite the homogenizing effect of pollen gene flow known for this species, the genetic structure should be taken into account in the management of the genetic resources of the common ash. Although ash dieback poses an additional challenge for the management of genetic resources, efforts should be directed towards protecting populations with high genetic diversity within defined phylogenetic units, as they may be an important source of adaptive variation for future stands.

Characterization, multivariate analysis and bioactivity evaluation of coumarins in the bark of Fraxinus mandshurica.

The bark of Fraxinus mandshurica is a traditional folk herb used to clear heat and dry dampness. To investigate the differences in coumarins content in the bark of F. mandshurica, 24 batches of samples from four origins were collected and analyzed. Eight coumarins were obtained by traditional natural product extraction, isolation and identification techniques and quantified by high performance liquid chromatography-photodiode array (HPLC-DAD). The quantitative results showed that the overall content of compound 30 (Fraxinol) was higher at 100.23 mg/g, while the overall content of compound 23 (Cichoriin) was lower, which may be related to environmental factors in different regions. The method validation showed that the linear range of the eight standards was between 10 and 2500 µg/mL with correlation coefficient (R2) values >0.9991; the relative standard deviation (RSD, %) values of intra-day precision were between 0.35 and 1.38, while the RSD values of inter-day precision were between 0. 29-1.78; the RSD (%) values for the reproducibility experiments ranged from 0.29 to 1.87, while the RSD (%) values for the stability experiments ranged from 0.22 to 2.33; the spiked recovery of the samples ranged from 98.65 to 101.34%, and the RSD (%) values ranged from 0.22 to 1.96. The method validation results showed that the instrument used for the analysis had good precision, the reproducibility and stability of the samples were good, and the accuracy of the experimental method was high. In addition, a total of 54 chemical components were identified from F. mandshurica bark by ultra performance liquid chromatography-electrospray quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS). Based on this, fingerprinting, heatmap and multivariate analysis, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were established for 24 batches of samples, and four marker compounds that could be used to distinguish different origins of F. mandshurica were screened. To further investigate the bioactivities of the eight coumarins, in vitro enzyme activity inhibition studies were performed, and the results showed that they all exhibited different degrees of inhibition of acetylcholinesterase, tyrosinase and α-glucosidase, thus having potential applications in the treatment of Alzheimer's disease, blemish whitening and anti-diabetes, and becoming a new source of natural enzyme activity inhibitors. This study established an identification and evaluation method applicable to plants of different origins, which provides a strong reference for quality control, origin evaluation and clinical application of traditional medicinal plants.

The mitochondrial genome and life history of Tomostethus sinofraxini (Hymenoptera: Tenthredinidae), an emerging pest of Fraxinus chinensis.

Tomostethus sinofraxini Wang & Wei (a new name is proposed for Tomostethus fraxini Niu & Wei, 2022: Tomostethus sinofraxini Wang & Wei, nom. nov.), an emerging sawfly pest of the Chinese ash, Fraxinus chinensis, is now endemic to Beijing, Tianjin, Hebei, and Shandong provinces. Given the severity of its infestation and the speed of its range expansion, we studied the phylogenetic relationship of T. sinofraxini with other sawfly species and its life history to be better informed for the management strategies. The nearly complete T. sinofraxini mitogenome is 16,169 bp in length and encodes 2 ribosomal RNAs (rrnL and rrnS), 22 transfer RNAs (tRNAs), and 13 protein-coding genes. The nucleotide composition is biased toward adenine and thymine (A + T = 81.7%). In comparison to the architecture of the ancestral insect mitogenome, 2 transposition events occur on the IQM tRNA cluster, rearranging it from IQM to MQI. Our phylogenetic analysis suggests that T. sinofraxini belongs to a group composed of paraphyletic subfamilies Blennocampinae and Heterarthrinae. In addition, to document its life history, we observed T. sinofraxini development at 2 geographical locations in Beijing, China, with different altitudes. At Jiulong Mountain, with a higher altitude and a lower average temperature, the developmental time of egg, larval, and adult stages was 19%-31% longer than that observed at the Chinese Academy of Forestry. A basic understanding of biological traits and molecular signatures is the critical first step to develop an integrated pest management framework for this emerging pest of the Chinese ash.

High quality genomes produced from single MinION flow cells clarify polyploid and demographic histories of critically endangered Fraxinus (ash) species.

With populations of threatened and endangered species declining worldwide, efforts are being made to generate high quality genomic records of these species before they are lost forever. Here, we demonstrate that data from single Oxford Nanopore Technologies (ONT) MinION flow cells can, even in the absence of highly accurate short DNA-read polishing, produce high quality de novo plant genome assemblies adequate for downstream analyses, such as synteny and ploidy evaluations, paleodemographic analyses, and phylogenomics. This study focuses on three North American ash tree species in the genus Fraxinus (Oleaceae) that were recently added to the International Union for Conservation of Nature (IUCN) Red List as critically endangered. Our results support a hexaploidy event at the base of the Oleaceae as well as a subsequent whole genome duplication shared by Syringa, Osmanthus, Olea, and Fraxinus. Finally, we demonstrate the use of ONT long-read sequencing data to reveal patterns in demographic history.

Protective effect of a hydromethanolic extract from Fraxinus excelsior L. bark against a rat model of aluminum chloride-induced Alzheimer's disease: Relevance to its anti-inflammatory and antioxidant effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Fraxinus excelsior L. (FE), commonly known as the ash, belongs to the Oleaceae family and has shown several pharmacological and biological properties, such as antioxidant, immunomodulatory, neuroprotective, and anti-inflammatory effects. It has also attracted the most attention toward neuroinflammation. Moreover, FE bark and leaves have been used to treat neurological disorders, aging, neuropathic pain, urinary complaints, and articular pain in traditional and ethnomedicine. Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder resulting from the involvement of amyloid-beta, metal-induced oxidative stress, and neuroinflammation. AIM OF THE STUDY: The objective of the current study was to assess the neuroprotective effects of hydromethanolic extract from FE bark in an AlCl3-induced rat model of AD. MATERIALS AND METHODS: The maceration process was utilized to prepare the hydromethanolic extract of FE bark, and characterized by LC-MS/MS. To assess the anti-AD effects of the FE extract, rats were categorized into five different groups, AlCl3; normal control; FE-treated groups at 50, 100, and 200 mg/kg. Passive avoidance learning test, Y-maze, open field, and elevated plus maze behavioral tests were evaluated on days 7 and 14 to analyze the cognitive impairments. Zymography analysis, biochemical tests, and histopathological changes were also followed in different groups. RESULTS: LC-MS/MS analysis indicated the presence of coumarins, including isofraxidin7-O-diglucoside in the methanolic extract of FE as a new isofraxidin derivative in this genus. FE significantly improved memory and cognitive function, maintained weight, prevented neuronal damages, and preserved the hippocampus's histological features, as demonstrated by behavioral tests and histopathological analysis. FE increased anti-inflammatory MMP-2 activity, whereas it decreased that of inflammatory MMP-9. Moreover, FE increased plasma antioxidant capacity by enhancing CAT and GSH while decreasing nitrite levels in the serum of treated groups. In comparison between the treated groups, the rats that received high doses of the FE extract (200 mg/kg) showed the highest therapeutic effect. CONCLUSION: FE rich in coumarins could be an effective anti-AD adjunct agent, passing through antioxidant and anti-inflammatory pathways. These results encourage further studies for the development of this extract as a promising agent in preventing, managing, or treating AD and related diseases.

Testing the heat treatment dose for Agrilus planipennis (Coleoptera: Buprestidae) prepupae using the Humble water bath.

The lethal heat treatment dose (time and temperature) for the prepupal life stage of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), emerald ash borer (EAB), was determined through an in vitro application using a carefully calibrated heat treatment apparatus. The lethal and sublethal effects of heat on A. planipennis prepupae were assessed through a ramped heat delivery application, simulating industrial kilns and conventional heat chamber operations, for treatments combining target temperatures of 54 °C, 55 °C, and 56 °C, and exposure durations of 0 min (i.e., kiln temperature ramp only), 15 min, or 30 min. Prepupal EAB larvae did not survive exposure to 56 °C for 15 min or longer, or to 55 °C for 30 min. Sublethal effects were observed for all other treatments. Sublethal effects included delayed development and failure to complete the pupal and adult life stages.

Emerald Ash Borer Management and Research: Decades of Damage and Still Expanding.

Since the discovery of the ash tree (Fraxinus spp.) killer emerald ash borer (EAB; Agrilus planipennis) in the United States in 2002 and Moscow, Russia in 2003, substantial detection and management efforts have been applied to contain and monitor its spread and mitigate impacts. Despite these efforts, the pest continues to spread within North America. It has spread to European Russia and Ukraine and is causing sporadic outbreaks in its native range in China. The dynamics of EAB's range expansion events appear to be linked to the lack of resistant ash trees in invaded ranges, facilitated by the abundance of native or planted North American susceptible ash species. We review recently gained knowledge of the range expansion of EAB; its ecological, economic, and social impacts; and past management efforts with their successes and limitations. We also highlight advances in biological control, mechanisms of ash resistance, and new detection and management approaches under development, with the aim of guiding more effective management.

Parasitoid-induced changes in metabolic rate and feeding activity of the emerald ash borer (Coleoptera: Buprestidae): implications for biological control.

Parasitoid-host interactions form the foundation of biological control strategies against many agriculture and forest insect pests. The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is a serious invasive pest of ash (Fraxinus spp.) trees in North America. Tetrastichus planipennisi (Hymenoptera: Eulophidae) is a gregarious, koinobiont endoparasitoid, attacking late (3rd to 4th) instars of EAB larvae, which feed in the live phloem of ash trunks or branches, making serpentine-like galleries filled with larval frass. In the present study, we tested the hypothesis that T. planipennisi regulates the host metabolism and feeding activity to optimize its offspring development and fitness. We first compared the respiration rate of parasitized and unparasitized host larvae at different times after parasitism, and then measured feeding activity of both parasitized and unparasitized host larvae inside their feeding galleries. Although parasitized host larvae increased metabolic rate and feeding activity in the first few days of parasitism, T. planipennisi parasitism induced an overall reduction of the metabolic rate and decrease in feeding activity of parasitized host larvae over their development period. In addition, there was a negative relationship between feeding activity of parasitized hosts and brood sizes of the parasitoid progeny-i.e., the more parasitoid progeny a host larva received, the less feeding activity the host had. These findings suggest that T. planipennisi has limited ability to optimize its offspring development and fitness through regulations of the host metabolism and feeding activity and its parasitism reduces feeding damage of parasitized EAB larvae to infested ash trees.

The chromosome-level genome assembly of Fraxinus americana provides insights into the evolution of Oleaceae plants.

White ash (Fraxinus americana linn.) originates from the southeastern United States. It is a tall and fast-growing tree species with strong salt-alkali resistance and cold tolerance, making it an important reforestation species and widely planted worldwide. Here, we completed the chromosome-level reference genome assembly of F. americana based on Illumina, PacBio, and Hi-C reads, with a genome size of 878.98 Mb, an N50 of 3.27 Mb, and a heterozygosity rate of 0.3 %. Based on de novo prediction, transcriptome prediction, and homology-based protein prediction, we obtained 39,538 genes. Approximately 843.21 Mb of the assembly genome was composed of 37,928 annotated protein-coding genes, with a gene function annotation rate of 95.93 %. 99.94 % of the overlap clusters (877.44 Mb) were anchored to 23 chromosomes. Synteny analysis of F. americana and other Oleaceae plants showed that F. americana underwent frequent chromosome rearrangements. The amplification of the Ale transposons effectively promoted the genome size of F. americana. Compared with other Oleaceae plants, the Glutathione S-transferase (GST) gene family in the F. americana genome has undergone significant expansion, which may help F. americana cope with adverse natural environments. Furthermore, we found that key enzyme-coding gene families related to lignin biosynthesis were expanded and highly expressed in F. americana leaves. These key genes drive lignin synthesis and benefit F. americana in fast-growing, as well as resisting biotic and abiotic stress. Overall, the F. americana genome assembly provides insights into the evolution of Oleaceae plants and provides abundant resources for breeding and germplasm conservation of white ash.

Transcriptome profiling of Fraxinus excelsior genotypes infested by emerald ash borer.

European ash, Fraxinus excelsior is facing the double threat of ongoing devastation by the invasive fungal pathogen, Hymenoscyphus fraxineus and the imminent arrival of the non-native emerald ash borer (EAB), Agrilus planipennis. The spread of EAB which is currently moving westwards from European Russia and Ukraine into central Europe, poses an additional substantial threat to European ash, F. excelsior. While the molecular basis for resistance or variation in resistance among European ash genotypes is heavily investigated, comparatively little is known about the molecular ash traits involved in resistance against EAB. In this study we have gathered transcriptomic data from EAB inoculated genotypes of F. excelsior that have previously shown different levels of susceptibility to EAB. Resultant datasets show differential gene expression in susceptible and resistant genotypes in response to EAB infestation. This data will provide important information on the molecular basis of resistance to the EAB and allow the development of management plans to combat a pending threat of a culturally and ecologically important European tree species.

Alien eating alien - rapid spread of Aceria fraxiniflora, a non-native gall mite of the invasive green ash (Fraxinus pennsylvanica) in Central-Eastern Europe.

The North American gall mite Aceria fraxiniflora was first recorded in Europe in southeast Hungary in 2017. Since then, it has shown a remarkably rapid spread on its host, the also North American green ash (Fraxinus pennsylvanica). By the beginning of 2023 it has been recorded in eight Central-Eastern European countries. In 2022 it was recorded on the other North American ash (Fraxinus Americana) in Zagreb (Croatia) and in Szarvas Arboretum (SE Hungary). Possible reasons and outcomes of this spread are discussed.

Successful establishment, spread, and impact of the introduced parasitoid Spathius galinae (Hymenoptera: Braconidae) on emerald ash borer (Coleoptera: Buprestidae) populations in postinvasion forests in Michigan.

Spathius galinae is a larval parasitoid native to the Russian Far East that was approved for release in the United States in 2015 for biological control of the emerald ash borer (EAB), Agrilus planipennis, an invasive beetle from Asia responsible for widespread mortality of ash trees (Fraxinus spp.) in North America. From 2015 to 2017, 1,340-1,445 females of S. galinae along with males were released into each release plot, paired with a nonrelease control plot (1-12.5 km apart), at 6 postinvasion forested sites containing abundant pole-sized ash trees in Michigan. By 2018, S. galinae had spread to all but one control plot. Based on the first year that S. galinae was found in trees in each control plot and the distances of those trees to the parasitoid release point within each site, we estimated that S. galinae spread at 3.7 (±1.9) km per year after its initial releases in 2015. The proportion of sampled trees with S. galinae broods, brood densities within sampled trees, and parasitism of EAB larvae increased sharply in both control and release plots after the last field releases in 2017, with the highest parasitism rates (42.8-60.3%) in 2020. Life table analysis showed that S. galinae alone reduced EAB's net population growth rate by 35-55% across sites from 2018 to 2020. These results demonstrate that S. galinae has established an increasing population in Michigan and now plays a significant role in reducing EAB populations in the area.

A glimmer of hope - ash genotypes with increased resistance to ash dieback pathogen show cross-resistance to emerald ash borer.

Plants rely on cross-resistance traits to defend against multiple, phylogenetically distinct enemies. These traits are often the result of long co-evolutionary histories. Biological invasions can force naïve plants to cope with novel, coincident pests, and pathogens. For example, European ash (Fraxinus excelsior) is substantially threatened by the emerald ash borer (EAB), Agrilus planipennis, a wood-boring beetle, and the ash dieback (ADB) pathogen, Hymenoscyphus fraxineus. Yet, plant cross-resistance traits against novel enemies are poorly explored and it is unknown whether naïve ash trees can defend against novel enemy complexes via cross-resistance mechanisms. To gain mechanistic insights, we quantified EAB performance on grafted replicates of ash genotypes varying in ADB resistance and characterized ash phloem chemistry with targeted and untargeted metabolomics. Emerald ash borer performed better on ADB-susceptible than on ADB-resistant genotypes. Moreover, changes in EAB performance aligned with differences in phloem chemical profiles between ADB-susceptible and ADB-resistant genotypes. We show that intraspecific variation in phloem chemistry in European ash can confer increased cross-resistance to invasive antagonists from different taxonomic kingdoms. Our study suggests that promotion of ADB-resistant ash genotypes may simultaneously help to control the ADB disease and reduce EAB-caused ash losses, which may be critical for the long-term stability of this keystone tree species.

Effects of exogenous calcium on growth, chlorophyll fluorescence characteristics and antioxidant system of Fraxinus malacophylla seedlings.

Karst ecosystems are becoming increasingly problematic, and high calcium is one of the main characteristics of soils in rocky desertification areas. Chlorophyll fluorescence is one of the most important indicators of the extent to which plants are affected by their environment. There are few reports on the effects of changes in exogenous calcium levels on the chlorophyll fluorescence properties of Fraxinus malacophylla seedlings. In the present study, we investigated the growth, chlorophyll fluorescence properties and antioxidant system of Fraxinus malacophylla seedlings in response to exogenous calcium (as the concentrations of 0, 25, 50, 75 mmol L-1). The results showed that Ca2+ concentration (25-50 mmol L-1) treatment mainly promoted the growth, biomass accumulation, root activity, and chlorophyll synthesis and effect on chlorophyll fluorescence in Fraxinus malacophylla; the developed root system became a strong linking hub for calcium adaptation. In addition, the activities of the antioxidant enzymes peroxidase (POD) and catalase (CAT) are upregulated and play an important role in preventing excessive oxidative damage. OJIP test parameters changed significantly with the addition of exogenous calcium, and parameters related to each photosystem II (PSII) reaction centre, such as ABS/RC and DIo/RC, increased significantly in the OJIP test, with enhanced function of the PSII electron donor lateral oxygen evolution complex. In conclusion, the addition of exogenous calcium (25-50 mmol L-1) had an important protective effect on the photosynthetic mechanism of Fraxinus malacophylla, promoting photosynthesis, better growth and better adaptability.

Establishment and impacts of emerald ash borer (Coleoptera: Buprestidae) parasitoids released at early- and post-invasion sites.

Forest stands infested by emerald ash borer (EAB), Agrilus planipennis Fairmaire, experience extensive mortality of mature ash trees. Post-invasion woodlands commonly have a small contingent of mature lingering ash, an orphaned cohort of seedlings/saplings, and low EAB densities. To protect regenerating ash against rebounding EAB populations, a suite of biocontrol agents are being reared and released. USDA APHIS guidelines currently recommend the release of parasitoids into forests prior to overstory ash mortality at sites containing a variety of ash size classes and low to moderate but building EAB densities. To understand if biocontrol establishment and control of EAB is feasible in post-invasion sites, we assessed the establishment of parasitoids in 6 post-invasion forest stands in 2 regions of NY and compared EAB mortality in these stands to 2 regions where releases were conducted during the early-invasion phase. Results of parasitoid trapping indicates Tetrastichus planipennisi Yang established under both release strategies. Spathius galinae Belokobylskij & Strazanac was only released in post-invasion stands, where it was established successfully. Artificial EAB cohorts were established and life tables were constructed at 3 sites per region. EAB mortality due to T. planipennisi parasitism was similar under both release strategies 2 yr after release in post-invasion stands versus 8 yr after release in early-invasion stands. Combined mortality from T. planipennisi and woodpecker predation resulted in consistently low EAB reproductive rates. Future biocontrol releases could target forests identified as economically or ecologically important, regardless of whether EAB populations are increasing or have collapsed following initial invasion.

Postrelease assessment of Oobius agrili (Hymenoptera: Encyrtidae) establishment and persistence in Michigan and the Northeastern United States.

The emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is an invasive woodboring pest of ash trees (Fraxinus sp.) in North America. Among the Asiatic parasitoids being released for the management of EAB in North America, Oobius agrili Zhang and Huang (Hymenoptera: Encyrtidae) is the only EAB egg parasitoid. To date, more than 2.5 million O. agrili have been released in North America; however, few studies have examined its success as a biological control agent of EAB. We conducted studies to assess O. agrili establishment, persistence, spread, and EAB egg parasitism rates in Michigan at the earliest release sites (2007-2010), as well as at more recent release sites (2015-2016) in 3 Northeastern United States (Connecticut, Massachusetts, New York). In both regions, we documented successful O. agrili establishment at all but one release site. In Michigan, O. agrili has persisted at release sites for over a decade and spread to all control sites located 0.6-3.8 km from release sites. Overall, EAB egg parasitism ranged from 1.5% to 51.2% (mean of 21.4%) during 2016-2020 in Michigan and from 2.6% to 29.2% (mean of 16.1%) during 2018-2020 in the Northeastern states. Future research efforts should focus on factors affecting the spatiotemporal variation in EAB egg parasitism rates by O. agrili, as well as its potential range in North America.

A comprehensive analysis of the chemical composition of Fraxinus mandshurica roots based on HPLC-DAD-MS, GC-MS and UPLC-ESI-Q-TOF-MS.

Fraxinus mandshurica is widely distributed around the world, which has significant economic value and pharmacological effects. However, its roots are often overlooked during processing and use. In this study, the preliminary identification of chemical components in F. mandshurica roots was carried out using ultra-performance liquid chromatography coupled with electrospray quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS), and a total of 37 characteristic components were identified, including 13 secoiridoids, 7 lignans, 6 coumarins, 4 flavonoids, 4 phenylethanols, 1 terpenoid and 2 other compounds. Meanwhile, to evaluate the 6 lignans content of F. mandshurica roots, the identification, quantitation analysis and methodological validation were performed by high-performance liquid chromatography with diode array detection mass spectrometry (HPLC-DAD-MS), which showed that the concentration range of standard compounds was 0.5-1000 µg/mL. The standard curve correlation coefficients (R2) were all greater than 0.9991, indicating that the linearity of the fitted curves was good. The highest lignan content in F. mandshurica roots was olivil (461.11 µg/g) and the lowest in buddlenol E (11.14 µg/g), and the total lignan content was 764.63 µg/g. The relative standard deviations (RSD, %) of both intra-day and inter-day precision were less than 1.95%. The RSD (%) of the reproducibility and stability experiments was less than 2.91%. The spiked recoveries of the samples were in the range of 98.29-102.62%, and the RSD (%) was in the range of 0.43-1.73, indicating that the method has high accuracy. In addition, a total of 20 volatile components in F. mandshurica roots were identified and quantified by gas chromatography-mass spectrometer (GC-MS) in this study, which laid a solid foundation for the comprehensive development and utilization of F. mandshurica resources.

Transcriptomic and metabolomic analysis of autumn leaf color change in Fraxinus angustifolia.

Fraxinus angustifolia is a type of street tree and shade tree with ornamental value. It has a beautiful shape and yellow or reddish purple autumn leaves, but its leaf color formation mechanism and molecular regulation network need to be studied. In this study, we integrated the metabolomes and transcriptomes of stage 1 (green leaf) and stage 2 (red-purple leaf) leaves at two different developmental stages to screen differential candidate genes and metabolites related to leaf color variation. The results of stage 1 and stage 2 transcriptome analysis showed that a total of 5,827 genes were differentially expressed, including 2,249 upregulated genes and 3,578 downregulated genes. Through functional enrichment analysis of differentially expressed genes, we found that they were involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, pigment metabolism, carotene metabolism, terpenoid biosynthesis, secondary metabolite biosynthesis, pigment accumulation, and other biological processes. By measuring the metabolites of Fraxinus angustifolia leaves, we found the metabolites closely related to the differentially expressed genes in two different periods of Fraxinus angustifolia, among which flavonoid compounds were the main differential metabolites. Through transcriptome and metabolomics data association analysis, we screened nine differentially expressed genes related to anthocyanins. Transcriptome and qRT-PCR results showed that these nine genes showed significant expression differences in different stages of the sample, and we speculate that they are likely to be the main regulatory factors in the molecular mechanism of leaf coloration. This is the first time that we have analyzed the transcriptome combination metabolome in the process of leaf coloration of Fraxinus angustifolia, which has important guiding significance for directional breeding of colored-leaf Fraxinus species and will also give new insights for enriching the landscape.

Gut Bacteria of Wood-Boring Streltzoviella insularis and Their Involvement in Pinoresinol Degradation of Ash Tree (Fraxinus sp.) Host.

Insect gut microbiota have been widely reported to help the insects to overcome host tree defense. Streltzoviella insularis (Lepidoptera: Cossidae) is one of the most common wood borers in China, attacking various hosts, including ash trees (Fraxinus sp.), but little is known about its gut microbial associates and their involvement in host tree defense. We isolated gut bacteria of S. insularis larvae, analyzed their ability to degrade pinoresinol (a defense compound of ash trees) and cellulose, and identified pinoresinol degradation products. Larval mortality increased with increasing pinoresinol concentration (reflecting natural variation observed in the host trees). All the five detected gut bacteria isolates were able to degrade pinoresinol, two of which were also capable of cellulose degradation. Furthermore, gut bacteria were also shown to degrade pinoresinol via the gluconeogenesis pathway. These results suggest that S. insularis-associated microorganisms help to overcome host pinoresinol defense and possibly contribute to insects or gut microbial nutrition via carbohydrate synthesis.