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.

Strong antagonism of an endophyte of Fraxinus excelsior towards the ash dieback pathogen, Hymenoscyphus fraxineus, is mediated by the antifungal secondary metabolite PF1140.

Ash dieback, caused by the fungal pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), is threatening the existence of the European ash, Fraxineus excelsior. During our search for biological control agents for this devastating disease, endophytic fungi were isolated from healthy plant tissues and co-cultivated with H. fraxineus to assess their antagonistic potential. Among the strains screened, Penicillium cf. manginii DSM 104493 most strongly inhibited the pathogen. Initially, DSM 104493 showed promise in planta as a biocontrol agent. Inoculation of DSM 104493 into axenically cultured ash seedlings greatly decreased the development of disease symptoms in seedlings infected with H. fraxineus. The fungus was thus cultivated on a larger scale in order to obtain sufficient material to identify active metabolites that accounted for the antibiosis observed in dual culture. We isolated PF1140 (1) and identified it as the main active compound in the course of a bioassay-guided isolation strategy. Furthermore, its derivative 2, the mycotoxin citreoviridin (3), three tetramic acids of the vancouverone type (4-6), and penidiamide (7) were isolated by preparative chromatography. The structures were elucidated mainly by NMR spectroscopy and high-resolution mass spectrometry (HRMS), of which compounds 2 and 6 represent novel natural products. Of the compounds tested, not only PF1140 (1) strongly inhibited H. fraxineus in an agar diffusion assay but also showed phytotoxic effects in a leaf puncture assay. Unfortunately, both the latent virulent attributes of DSM 104493 observed subsequent to these experiments in planta and the production of mycotoxins exclude strain Penicillium cf. manginii DSM 104493 from further development as a safe biocontrol agent.IMPORTANCEEnvironmentally friendly measures are urgently needed to control the causative agent of ash dieback, Hymenoscyphus fraxineus. Herein, we show that the endophyte DSM 104493 exhibits protective effects in vitro and in planta. We traced the activity of DSM 104493 to the antifungal natural product PF1140, which unfortunately also showed phytotoxic effects. Our results have important implications for understanding plant-fungal interactions mediated by secondary metabolites, not only in the context of ash dieback but also generally in plant-microbial interactions.

Distinct metabolites affect the phloem fungal communities in ash trees (Fraxinus spp.) native and nonnative to the highly invasive emerald ash borer (AGRILUS PLANIPENNIS).

Emerald ash borer (EAB, Agrilus planipennis) is an invasive killer of ash trees (Fraxinus spp.) in North America and Europe. Ash species co-evolved with EAB in their native range in Asia are mostly resistant, although the precise mechanism(s) remain unclear. Very little is also known about EAB or ash tree microbiomes. We performed the first joint comparison of phloem mycobiome and metabolites between a native and a nonnative ash species, infested and uninfested with EAB, in conjunction with investigation of larval mycobiome. Phloem mycobiome communities differed between the tree species, but both were unaffected by EAB infestation. Several indicator taxa in the larval gut shared a similarly high relative abundance only with the native host trees. Widely targeted metabolomics revealed 24 distinct metabolites in native trees and 53 metabolites in nonnative trees, respectively, that differed in relative content between infested and uninfested trees only in one species. Interestingly, four metabolites shared a strong relationship with the phloem mycobiomes, majority of which affected only the native trees. Collectively, our results demonstrate a complex interplay between host tree chemistry and mycobiome, and suggest the shared relationships between the mycobiomes of the native host tree and EAB may reflect their shared co-evolution.

Possible Biological Control of Ash Dieback Using the Mycoparasite Hymenoscyphus Fraxineus Mitovirus 2.

Invasive fungal diseases represent a major threat to forest ecosystems worldwide. As the application of fungicides is often unfeasible and not a sustainable solution, only a few other control options are available, including biological control. In this context, the use of parasitic mycoviruses as biocontrol agents of fungal pathogens has recently gained particular attention. Since the 1990s, the Asian fungus Hymenoscyphus fraxineus has been causing lethal ash dieback across Europe. In the present study, we investigated the biocontrol potential of the mitovirus Hymenoscyphus fraxineus mitovirus 2 (HfMV2) previously identified in Japanese populations of the pathogen. HfMV2 could be successfully introduced via co-culturing into 16 of 105 HfMV2-free isolates. Infection with HfMV2 had contrasting effects on fungal growth in vitro, from cryptic to detrimental or beneficial. Virus-infected H. fraxineus isolates whose growth was reduced by HfMV2 showed overall a lower virulence on ash (Fraxinus excelsior) saplings as compared with their isogenic HfMV2-free lines. The results suggest that mycoviruses exist in the native populations of H. fraxineus in Asia that have the potential for biological control of ash dieback in Europe. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

Traces of Hymenoscyphus fraxineus in Northeastern Europe Extend Further Back in History than Expected.

Herbaria are a promising but still poorly applied information source for retrospective microbiological studies. In order to find any evidence of the virulent European origin of ash dieback agent Hymenoscyphus fraxineus and other fungal pathogens, we analyzed 109 leaf samples from three different Estonian botanical herbaria, sampled during 171 years from 20 ash species and cultivars, using a PacBio third-generation sequencing of the fungal internal transcribed spacer ITS1-5.8S-ITS2 ribosomal DNA region. We identified a large amount of saprotrophic fungi naturally colonizing ash leaves. Hymenoscyphus fraxineus colonized a Fraxinus chinensis subsp. rhynchophylla specimen and a F. chinensis specimen collected from Tallinn Botanic Garden in July 1978 and July 1992, respectively. The samples originated from trees grown in this garden from seeds collected from Shamora, Far-East Russia, in 1961 and from a Beijing botanical garden in eastern China in 1985, respectively. Repeated subsequent DNA extraction, real-time quantitative PCR, and Sanger and Illumina sequencing confirmed our findings of these apparently oldest cases of the ash dieback agent in Europe. These results show that H. fraxineus evidently was present in Estonia 19 years earlier than our previous data from fungal herbaria documented and 14 years before the first visible damage of ash trees was registered in Poland. Because we found no evidence of the saprotrophic H. albidus from earlier mycological and botanical herbarium specimens, the presence of H. albidus in Estonia remains questionable.

Meteorological factors associated with the timing and abundance of Hymenoscyphus fraxineus spore release.

The ascomycete Hymenoscyphus fraxineus has spread across most of the host range of European ash with a high level of mortality, causing important economic, cultural and environmental effects. We present a novel method combining a Monte-Carlo approach with a generalised additive model that confirms the importance of meteorology to the magnitude and timing of H. fraxineus spore emissions. The variability in model selection and the relative degree to which our models are over- or under-fitting the data has been quantified. We find that both the daily magnitude and timing of spore emissions are affected by meteorology during and prior to the spore emission diurnal peak. We found the daily emission magnitude has the strongest associations to weekly average net radiation and leaf moisture before the emission, soil temperature during the day before emission and net radiation during the spore emission. The timing of the daily peak in spore emissions has the strongest associations to net radiation both during spore emission and in the day preceding the emission. The seasonal peak in spore emissions has a near-exponential increase/decrease, and the mean daily emission peak is approximately Gaussian.

Transcriptional responses in developing lesions of European common ash (Fraxinus excelsior) reveal genes responding to infection by Hymenoscyphus fraxineus.

BACKGROUND: With the expanding ash dieback epidemic that has spread across the European continent, an improved functional understanding of the disease development in afflicted hosts is needed. The study investigated whether differences in necrosis extension between common ash (Fraxinus excelsior) trees with different levels of susceptibility to the fungus Hymenoscyphus fraxineus are associated with, and can be explained by, the differences in gene expression patterns. We inoculated seemingly healthy branches of each of two resistant and susceptible ash genotypes with H. fraxineus grown in a common garden. RESULTS: Ten months after the inoculation, the length of necrosis on the resistant genotypes were shorter than on the susceptible genotypes. RNA sequencing of bark samples collected at the border of necrotic lesions and from healthy tissues distal to the lesion revealed relatively limited differences in gene expression patterns between susceptible and resistant genotypes. At the necrosis front, only 138 transcripts were differentially expressed between the genotype categories while 1082 were differentially expressed in distal, non-symptomatic tissues. Among these differentially expressed genes, several genes in the mevalonate (MVA) and iridoid pathways were found to be co-regulated, possibly indicating increased fluxes through these pathways in response to H. fraxineus. Comparison of transcriptional responses of symptomatic and non-symptomatic ash in a controlled greenhouse experiment revealed a relatively small set of genes that were differentially and concordantly expressed in both studies. This gene-set included the rate-limiting enzyme in the MVA pathway and a number of transcription factors. Furthermore, several of the concordantly expressed candidate genes show significant similarity to genes encoding players in the abscisic acid- or Jasmonate-signalling pathways. CONCLUSIONS: A set of candidate genes, concordantly expressed between field and greenhouse experiments, was identified. The candidates are associated with hormone signalling and specialized metabolite biosynthesis pathways indicating the involvement of these pathways in the response of the host to infection by H. fraxineus.

Diversity of the 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma fraxini' isolates that infect urban trees in Bogotá, Colombia.

Phytoplasmas have been associated with a disease that affects trees of at least 11 species from different botanic families in Bogotá, Colombia. 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma fraxini' are the major groups of phytoplasma in the area of Bogotá. In this study, the genetic diversity within 'Ca. P. asteris' and 'Ca. P. fraxini' was studied in five urban tree species: Croton species (Euphorbiaceae), Fraxinus uhdei (Oleaceae), Magnolia grandiflora (Magnoliaceae), Populus nigra (Salicaceae) and Quercus humboldtii (Fagaceae). Analyses of the 16S rRNA gene using nested PCR, RFLP and sequencing showed that phytoplasmas of 'Ca. P. asteris' could be assigned to: subgroup 16SrI-B; a new subgroup named 16SrI-AF, with a restriction pattern similar to that of 16SrI-B; and a new subgroup named 16SrI-AG, with a restriction pattern similar to that of 16SrI-K and 16SrI-AH with a restriction pattern similar to that of 16SrI-AC. 'Ca. P. fraxini' isolates belonged to a new subgroup named 16SrVII-G, with a restriction pattern similar to that of 16SrVII-A. To complement the identification of the phytoplasma strains, we amplified nonribosomal genes such as leuS and secA. Unexpectedly, it was observed that in 16 trees in which 16S rRNA gene analysis showed the presence of 'Ca. P. fraxini' only, the leuS or secA primers amplified sequences exclusively affiliated to 'Ca. P. asteris. In those plants, sequences belonging to 'Ca. P. fraxini' leuS or secA genes were not amplified. The present work contributes to the identification of novel strains of both species in Colombia, and supports previous suggestions that phytoplasmas in South America are highly variable.

Leaf litter species identity influences biochemical composition of ectomycorrhizal fungi.

In forest ecosystems, ectomycorrhizal (ECM) fungi are important for plant growth and soil biogeochemical processes. The biochemical composition of ECM mycelium is an important fungal effect trait with consequences for its decomposition rate, and consequently on soil carbon pools and plant nutrition. Although the link between ECM fungi and leaf litter-released nutrients is well known, the response of ECM fungal biochemical composition to different leaf litter species remains poorly understood. To determine how leaf litter quality influences ECM fungi's biochemical profiles, we planted young beech trees in an oak forest and replaced the natural leaf litter with that of European beech (Fagus sylvatica), ash (Fraxinus excelsior), maple (Acer pseudoplatanus), or lime (Tilia cordata). We assessed the biochemical profiles of ECM root tips colonized by common fungal taxa in temperate forests (i.e., Cenococcum geophilum, Inocybe sp., and Lactarius subdulcis), using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). ECM fungal biochemical composition changed with leaf litter species. Changes were apparent in the infrared absorption bands assigned to functional groups of lipids, amides, and carbohydrates. C. geophilum and L. subdulcis exhibited large spectral differences corresponding to the initial pattern of leaf litter chemical composition between samples collected in the beech and ash leaf litter treatments. In contrast, Inocybe sp. was influenced by lime, but with no differences between samples from ash or beech leaf litter treatments. Although the spectral bands affected by leaf litter type differed among ECM fungi, they were mainly related to amides, indicating a dynamic response of the fungal proteome to soil nutritional changes. Overall, the results indicate that the biochemical response of ECM fungi to leaf litter species varies among ECM fungal species and suggests that the biochemical composition of ECM mycelium is a fungal response trait, sensitive to environmental changes such as shifts in leaf litter species.

Fungal diversity and seasonal succession in ash leaves infected by the invasive ascomycete Hymenoscyphus fraxineus.

High biodiversity is regarded as a barrier against biological invasions. We hypothesized that the invasion success of the pathogenic ascomycete Hymenoscyphus fraxineus threatening common ash in Europe relates to differences in dispersal and colonization success between the invader and the diverse native competitors. Ash leaf mycobiome was monitored by high-throughput sequencing of the fungal internal transcribed spacer region (ITS) and quantitative PCR profiling of H. fraxineus DNA. Initiation of ascospore production by H. fraxineus after overwintering was followed by pathogen accumulation in asymptomatic leaves. The induction of necrotic leaf lesions coincided with escalation of H. fraxineus DNA levels and changes in proportion of biotrophs, followed by an increase of ubiquitous endophytes with pathogenic potential. H. fraxineus uses high propagule pressure to establish in leaves as quiescent thalli that switch to pathogenic mode once these thalli reach a certain threshold - the massive feedback from the saprophytic phase enables this fungus to challenge host defenses and the resident competitors in mid-season when their density in host tissues is still low. Despite the general correspondence between the ITS-1 and ITS-2 datasets, marker biases were observed, which suggests that multiple barcodes provide better overall representation of mycobiomes.

Population genetic analysis of a parasitic mycovirus to infer the invasion history of its fungal host.

Hymenoscyphus fraxineus mitovirus 1 (HfMV1) occurs in the fungus Hymenoscyphus fraxineus, an introduced plant pathogen responsible for the devastating ash dieback epidemic in Europe. Here, we explored the prevalence and genetic structure of HfMV1 to elucidate the invasion history of both the virus and the fungal host. A total of 1298 H. fraxineus isolates (181 from Japan and 1117 from Europe) were screened for the presence of this RNA virus and 301 virus-positive isolates subjected to partial sequence analysis of the viral RNA polymerase gene. Our results indicate a high mean prevalence (78.7%) of HfMV1 across European H. fraxineus isolates, which is supported by the observed high transmission rate (average 83.8%) of the mitovirus into sexual spores of its host. In accordance with an expected founder effect in the introduced population in Europe, only 1.1% of the Japanese isolates were tested virus positive. In Europe, HfMV1 shows low nucleotide diversity but a high number of haplotypes, which seem to be subject to strong purifying selection. Phylogenetic and clustering analysis detected two genetically distinct HfMV1 groups, both present throughout Europe. This pattern supports the hypothesis that only two (mitovirus-carrying) H. fraxineus individuals were introduced into Europe as previously suggested from the bi-allelic nature of the fungus. Moreover, our data points to reciprocal mating events between the two introduced individuals, which presumably initiated the ash dieback epidemic in Europe.

Rising Out of the Ashes: Additive Genetic Variation for Crown and Collar Resistance to Hymenoscyphus fraxineus in Fraxinus excelsior.

Since the early 1990s, ash dieback due to the invasive ascomycete Hymenoscyphus fraxineus is threatening Fraxinus excelsior in most of its natural range. Previous studies reported significant levels of genetic variability in susceptibility in F. excelsior either in field or inoculation experiments. The present study was based on a field experiment planted in 1995, 15 years before onset of the disease. Crown and collar status were monitored on 777 trees from 23 open-pollinated progenies originating from three French provenances. Health status was modeled using a Bayesian approach where spatiotemporal effects were explicitly taken into account. Moderate narrow-sense heritability was found for crown dieback (h2 = 0.42). This study is first to show that resistance at the collar level is also heritable (h2 = 0.49 for collar lesions prevalence and h2 = 0.42 for their severity) and that there is significant genetic correlation (r = 0.40) between the severities of crown and collar symptoms. There was no evidence for differences between provenances. Family effects were detected, but computing individual breeding values showed that most of the genetic variation lies within families. In agreement with previous reports, early flushing correlates with healthier crown. Implications of these results in disease management and breeding are discussed.

Pseudoxanthomonas humi sp. nov., a bacterium isolated from rhizospheric soil of Fraxinus chinensis in Gyeonggi Province, South Korea.

A novel bacterial strain THG-MM13(T) was isolated from rhizospheric soil sample and was characterized by using a polyphasic approach. Cells were Gram-reaction-negative, non-motile and rod-shaped. The strain was aerobic, catalase and oxidase positive, and optimum growth temperature and pH were 28 °C and 7.0, respectively. On the basis of 16S rRNA gene sequence analysis, strain THG-MM13(T) (KM598260) belongs to the genus Pseudoxanthomonas and is most closely related to Pseudoxanthomonas wuyuanensis KCTC 23877(T) (97.4 %) (JN247803), followed by Pseudoxanthomonas koreensis KCTC 12208(T) (96.7 %) (AY550263) and Pseudoxanthomonas yeongjuensis KACC 11580(T) (96.7 %) (DQ438977). The DNA G + C content was 63.7 mol%, and the predominant respiratory quinone was ubiquinone-8. The major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. The major fatty acids were iso-C15:0 (31.3 %) and iso-C16:0 (19.3 %). The DNA-DNA relatedness value between strain THG-MM13(T) and P. wuyuanensis KCTC 23877(T) was below 50 %. The DNA-DNA hybridization result and results of the genotypic analysis in combination with chemotaxonomic and physiological data demonstrated that strain THG-MM13(T) represented a novel species within the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas humi is proposed. The type strain is THG-MM13(T) (=KACC 18280(T) = CCTCC AB 2015122(T)).

[PATHOGENIC MIKO,- AND MICROFLORA OF FRAXINUS EXCELSIOR IN PODOLYA UKRAINE].

The article summarizes our research results of pathogenic myco- and microflora, as well as harmful entomofauna on European Ash. It is shown that the most common and harmful diseaseis tuberculosis (its causal agent--bacteria Pseudomonas syringae pv.savastanoi (Smith 1908), which affects trunks, branches, twigs and buds of European Ash. It describes a number of pathogens and representatives mikofitozov malicious entomofauna that by virtue of its activities significantly weaken the growth, development and underestimate the qualitative characteristics of wood European Ash.

Population structure of the invasive forest pathogen Hymenoscyphus pseudoalbidus.

Understanding the genetic diversity and structure of invasive pathogens in source and in introduced areas is crucial to the revelation of hidden biological features of an organism, to the reconstruction of the course of invasions and to the establishment of effective control measures. Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) is an invasive and highly destructive fungal pathogen found on common ash Fraxinus excelsior in Europe and is native to East Asia. To gain insights into its dispersal mechanisms and history of invasion, we used microsatellite markers and characterized the genetic structure and diversity of H. pseudoalbidus populations at three spatial levels: (i) between Europe and Japan, (ii) in Europe and (iii) at the epidemic's front in Switzerland. Phylogenetic and network analysis demonstrated that individuals from both regions are conspecific. However, populations from Japan harboured a higher genetic diversity and were genetically differentiated from European ones. No evident population structure was found among the 1208 European strains using Bayesian and multivariate clustering analysis. Only the distribution of genetic diversity in space, pairwise population differentiation (GST) and the spatial analysis of principal components revealed a faint geographical pattern around Europe. A significant allele deficiency in most European populations pointed to a recent genetic bottleneck, whereas no pattern of isolation by distance was found. Our data suggest that H. pseudoalbidus was introduced just once by at least two individuals. The potential source region of H. pseudoalbidus is vast, and further investigations are required for a more accurate localization of the source population.

A molecular tool for detection and tracking of a potential indigenous Beauveria bassiana strain for managing emerald ash borer populations in Canada.

Emerald ash borer is an invasive species from Asia. Beauveria bassiana strain L49-1AA is being tested for the control of emerald ash borer in Canada, using an autocontamination trapping system. We have developed a simplified allele discrimination polymerase chain reaction (PCR) assay to screen B. bassiana strain, L49-1AA from other Beauveria species by targeting the inter-strain genetic differences in 5' end of EF1-α gene of the genus Beauveria. A single nucleotide polymorphism (SNP) site, T→C was identified only in L49-1AA and was used to develop a simplified allele discrimination polymerase chain reaction (PCR) assay based on a modified allelic inhibition of displacement activity (AIDA) approach for distinguishing B. bassiana L49-1AA from all background Beauveria isolates. The SNP site was employed to design inner primers but with a deliberate mismatch introduced at the 3' antepenultimate from the mutation site in order to maximize specificity and detection efficiency. Amplification was specific to L49-1AA without cross-reaction with DNA from other Beauveria strains. In addition, the designed primers were also tested against environmental samples in L49-1AA released plots and observed to be highly efficient in detecting and discriminating the target strain, L49-1AA from both pure and crude DNA samples. This new method can potentially allow for more discriminatory tracking and monitoring of released L49-1AA in our autocontamination and dissemination projects for managing EAB populations. Additionally, the modified-AIDA format has potential as a tool for simultaneously identifying and differentiating closely related Beauveria species, strains/isolates as well as general classification of other pathogens or organisms.

A volatile lactone of Hymenoscyphus pseudoalbidus, pathogen of European ash dieback, inhibits host germination.

The largely unknown secondary metabolism of the plant pathogenic fungus Hymenoscyphus pseudoalbidus was investigated by use of the CLSA method. A set of volatile lactones was identified by GC/MS. The lactones were synthesized and used in bioassays in which one of the compounds was found to be a strong germination inhibitor for ash seeds, causing necroses in the plant tissue.