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Article

Detection of Viruses in Special Stands of Common Ash Reveals Insights into the Virome of Fraxinus excelsior

by
Marius Rehanek
*,
Rim Al Kubrusli
,
Kira Köpke
,
Susanne von Bargen
and
Carmen Büttner
Division Phytomedicine, Thaer-Institute of Agricultural and Horticultural Sciences, Humboldt-Universität zu Berlin, Lentzeallee 55/57, D-14195 Berlin, Germany
*
Author to whom correspondence should be addressed.
Forests 2024, 15(8), 1379; https://doi.org/10.3390/f15081379
Submission received: 9 July 2024 / Revised: 4 August 2024 / Accepted: 5 August 2024 / Published: 7 August 2024
(This article belongs to the Special Issue Forest Diseases and Pests: Recent Scientific Findings)
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Abstract

:
Plant diseases are mostly multicausal with several factors influencing the health status of affected hosts. Common ash (Fraxinus excelsior), a significant tree species of European forests, is currently mostly endangered by ash dieback, caused by the invasive fungus Hymenoscyphus fraxineus. However, contributing factors, including pathogenic viruses, are poorly understood. Here, we report the results of a virus screening conducted on selected special stands of F. excelsior. Over three consecutive years, ash trees from different origins were tested, including leaf material from mature seed trees, young trees and ash seedlings from the natural regeneration. Using RT-PCR, we screened for five viruses, including the generalist species ArMV (Nepovirus arabis) and CLRV (Nepovirus avii), as well as newly discovered viruses in ash, including the emaravirus ASaV (Emaravirus fraxini), the idaeovirus PrLBaV (Idaeovirus ligustri), and cytorhabdoviruses. The results revealed a high virus diversity in common ash. An association of ASaV detection with specific leaf symptoms, including shoestring, chlorotic ringspots, and vein yellowing, was documented. An analyses of relevant gene products of cytorhabdoviruses obtained from ashes of different sites revealed sequence diversities and two distinct phylogenetic groups present in ash populations. Signatures of novel viruses from different families have been identified by high-throughput sequencing. Together, our results provide insights into the virus diversity and distribution of viruses in ash and expand our knowledge about the virome of this endangered tree species.

1. Introduction

Virus infections of deciduous tree species have become more visible during the last two decades thanks to modern sequencing technologies, such as high-throughput sequencing (HTS), which has highlighted the high abundance, the evidence, and the diversity of viruses in forest sites [1]. Particularly, new members of the genus Emaravirus have recently been discovered in various forest tree species, including Eurasian aspen, sycamore maple, common oak, and common and flowering ash, raising them to a key virus genus in deciduous tree species [2]. As part of the phytobiome, viruses significantly contribute to the overall health status of trees, being responsible for economic and environmental losses [3]. The ability to rearrange host metabolism for the purpose of their own replication results in a loss of tree vitality, which alters its predisposition to other stress factors and pathogens [4]. Interactions of viruses with other abiotic and biotic factors occur [5].
Populations of common ash (Fraxinus excelsior L.) (F. excelsior) are currently severely damaged by ash dieback disease. As there are currently no means of combating the causative agent, the invasive ascomycete Hymenoscyphus fraxineus (H. fraxineus), the disease was able to spread unhindered throughout Europe during the last three decades, reaching Germany in 2002 [6]. In order to preserve common ash as an economically and ecologically important forest tree species, intense research has been initiated to investigate the pathogens’ epidemiology, to study systems and methodologies to control the spread of the disease and to develop breeding or silvicultural approaches [7]. However, investigations on further biotic and abiotic factors that shape the environment in which common ash is confronted with the fungal pathogen are missing. In this context, viruses must be considered as essential players. They can be transmitted in different ways, including vectors and seeds, which lead to a rapid distribution within a population [8]. Assessing the virus status of trees is crucial, as there is no possibility to cure virus-infected trees after infection. Special stands play a crucial role in biodiversity conservation, ecosystem management, and sustainable forest stewardship. Protecting and managing these areas is key for maintaining healthy forest ecosystems and ensuring their long-term viability [9]. In particular, studies on virus infections in special stands have not been carried out so far.
The multidisciplinary FraxVir project has been initiated to gain more knowledge about the virus status of F. excelsior. Viruses in trees have to be regarded as one of the strong influencing predisposing factors.
In the presented study, we provide results of our comprehensive survey for viruses that affect F. excelsior. The investigations comprised four main steps: 1. A visual inspection of the ash trees to document virus-suspected leaf symptoms. The sampling sites included five special stands in different regions of Germany, all of which are affected by ash dieback disease: two seed production sites, two seed orchards, and one clone archive (Table 1). 2. Sampling of leaf material. We collected leaves from ashes of different ages, including mature seed trees, young trees, and seedlings resulting from the reproduction of mature trees (natural regeneration). 3. Addressing the virus infection status of the trees by testing for different viruses. Generalist viruses known to affect ash were selected including arabis mosaic virus (ArMV, syn. Nepovirus arabis) [10] and cherry leaf roll virus (CLRV, syn. Nepovirus avii) [11]. These nepoviruses (order Picornavirales, family Secoviridae) are distributed worldwide. They have a wide host range, contain a bipartite genome, and are transmitted mechanically and through seeds [12]. In addition, we checked for recently discovered viruses in ash, for which little information is available so far. This includes ash shoestring-associated virus (ASaV, syn. Emaravirus fraxini) )), privet leaf blotch-associated virus (PrLBaV, syn. Idaeovirus ligustri))), and novel cytorhabdoviruses, which were identified by HTS in several Fraxinus spp. samples expressing virus-suspected leaf symptoms from locations in Germany (Emmendingen, Hamburg) and Norway (Ås) [13].
ASaV was first identified in diseased common ash trees from Switzerland [14]. It belongs to the genus Emaravirus (order Bunyavirales; family Fimoviridae), which contains segmented, single-stranded, negative-sense RNA viruses. Emaraviruses are worldwide distributed and cause significant diseases in economically and ecologically important host plants. They infect various host plant species and have especially become prevalent in important long-living woody plants, including rowan, oak, aspen, and sycamore maple. Emaraviruses are transmitted by eriophyid mites or by mechanical transmission. The segmented genome consists of four core genome segments encoding an RNA-dependent RNA polymerase (RNA 1), a glycoprotein precursor (RNA 2), a nucleocapsid protein (NC, RNA 3), and a movement protein (RNA 4) [2]. After its initial description [14], ASaV was also detected in manna ash (Fraxinus ornus) (F. ornus). The virus is distributed across European countries, including Germany, Switzerland, Italy, France, and Sweden [15].
The idaeovirus PrLBaV (order Martellivirales, family Mayoviridae) was first identified by HTS in privet (Ligustrum japonicum L.) showing leaf blotching. It belongs to the genus Idaeovirus and is composed of two single-stranded, positive-sense RNAs (RNA 1 and RNA 2). The phylogenetically closest related virus is raspberry bushy dwarf virus (RBDV), which occurs worldwide and has been frequently found infecting raspberries and blackberries [16]. Since RBDV is assumed to be transmitted by seed and pollen [16,17], this mode of spread should also be considered for PrLBaV. Ref. [16] also reported a partial PrLBaV sequence in the National Center for Biotechnology Information (NCBI) database, derived from a F. excelsior sample.
Members of the genus Cytorhabdovirus (order Mononegavirales, family Rhabdoviridae) have been detected in deciduous trees with conspicuous frequency. They are plant viruses that replicate in the cytoplasm of infected cells. They have a negative-sense, single-stranded RNA genome of 12.2–14.5 kb in size, comprising a conserved set of five canonical genes that encode a nucleocapsid protein (NC), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and a polymerase (L). Cytorhabdoviruses can infect a variety of host plants, including monocots and dicots and are transmitted by insects such as aphids, planthoppers, leafhoppers, and whiteflies. Mechanical transmission has also been demonstrated, as well as transmission during vegetative propagation [16]. Recently, novel viruses that possess characteristics typical of the genus have been identified in different host plants, including Tilia cordata [18]. In urban common and manna ash trees, we detected a so far unknown cytorhabdovirus by RT-PCR, which relates to findings from [19]. They assembled full-length sequences of two closely related species of cytorhabdoviruses (Fraxinus gammacytorhabdovirus 1, FraGCRV1 (BK064353.1); and Fraxinus gammacytorhabdovirus 2, FraGCRV2 (BK064354.1)) from published HTS datasets originating from F. excelsior [19]. The last step of our study was 4. Evaluating the virus diversity obtained by RT-PCR and identifying putative additional viruses by HTS analyses of selected trees.
The presented results expand the knowledge on the virome of ash and focuses on viruses as important factor contributing to ash health status.

2. Materials and Methods

In order to investigate the virus status of ash trees, we performed a visual inspection and sampled leaf material from F. excelsior belonging to different ages, comprising seedlings, 5–30 year-old young ashes and mature seed trees, 30 to approximately 130 years old. The virus status of each sample was assessed by an RT-PCR targeting different genomic regions. Additionally, an HTS was applied to identify currently unknown viruses.

2.1. Visual Inspection and Sampling of Plant Material

The visual inspection of ash trees to document virus-suspected leaf symptoms and the sampling of leaf material was conducted in two consecutive years, 2022 and 2023, from the below-mentioned sampling sites (Table 1).
The sites were chosen due to their special stand characters, such as seed plantations and seed production sites. We inspected the trees and collected leaves once in the period between June and August. Seedlings from Melzower Forst were additionally sampled in October 2021. The inspection comprised all ashes in the seed plantations Emmendingen (n = 140) and Schorndorf (n = 190) and the clone archive in Grabenstätt (n = 215). For the seed production sites Melzower Forst and Kaisheim, we focused on young ash trees and seedlings of the natural regeneration. The selected mature seed trees were investigated using a lift truck to reach the canopy and collect leaves.

2.2. RNA Isolation and cDNA Synthesis

Total RNA was extracted from 0.2 g fresh leaf material using a protocol modified by [20]. Following RNA isolation, cDNA was synthesized from 1 to 1.5 µg of total RNA using random hexamer oligonucleotides and Maxima H Minus Reverse Transcriptase (200 U/L) following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The quality of the cDNA was checked by amplification of the mitochondrial nad5 gene according to [21] as an internal control.

2.3. Virus Detection

The RT-PCR was performed in a 25 μL reaction mixture containing 2.5 μL 10× DreamTaq buffer (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 μL dNTPs (10 mM each), 0.25 μL forward and reverse primers (50 µM each) (Biolegio, Nijmegen, The Netherlands), 0.125 μL DreamTaq DNA polymerase (5 U/µL) (Thermo Fisher Scientific, Waltham, MA, USA), and 1 μL cDNA, adjusted to the final volume with DEPC-treated water. The cycling conditions were as follows: 94 °C for 3 min, and 35 cycles of 94 °C for 30 s, 52–58 °C for 30 s, and 72 °C for 20–50 s (adjusted to the fragment length generated by the primer sets), followed by 72 °C for 5 min. The primer pairs used in this study, their sequences, targeting regions, and annealing temperatures are given in the Supplementary Table S1.
PCR products (5 µL each) were examined by 1% agarose gel electrophoresis (Biozym LE agarose, Hessisch Oldendorf, Germany) with 1× TBE as electrophoresis buffer. The DNA was stained with Stain Clear G (Serva, Heidelberg, Germany) in a ratio of 1:10,000 and visualized using the E-Box CX5 (Vilber, Eberhardzell, Germany) according to the manufacturer’s instructions.

2.4. Sequencing

The selected PCR products were purified either directly using MSB Spin PCRapace Kit (Stratec SE, Birkenfeld, Germany) or by isolation from the agarose gel using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The purified DNA was sequenced by Sanger capillary sequencing (Macrogen-Europe, Amsterdam, The Netherlands) from both sides. For the evaluation, BioEdit Sequence Alignment Editor was used [22]. For phylogenetic analyses, we used Geneious Prime 2022.2.2 (Biomatters, Auckland, New Zealand), including the MUSCLE tool for the alignment of amino acid sequences [23]. Phylogenetic trees were generated using Geneious tree builder applying the Jukes–Cantor genetic distance model and the neighbor-joining method [24].

2.5. RNA-Seq Analyses

For the HTS, total RNA from ash leaves or male inflorescences were isolated as described above. For the RNA-Seq of leaf samples, RNA from 6 to 10 selected ash trees based on virus-suspected symptoms was isolated and pooled per sampling site. For RNA-Seq of male inflorescences, RNA from 10 randomly chosen ash trees was isolated and pooled per sampling site. We used 60 µg RNA per HTS sample. DNA was removed from the samples using DNase I (1 U/µL) according to the supplier’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). RNA was purified using the NucleoSpin® RNA Clean-up Kit (Macherey-Nagel, Düren, Germany) and precipitated in the presence of glycogen. Ribosomal RNA was depleted using the RiboMinus Plant Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Ribosomal RNA-depleted samples were used for cDNA synthesis with the MaximaH Minus double-stranded cDNA synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) primed with random hexamers according to the manufacturer’s protocol. Double-stranded (ds) cDNA was purified with the MSB Spin PCRapace Kit (Stratec SE, Birkenfeld, Germany). Samples were sent for library preparation (Nextera XT DNA Library Preparation Kit, Illumina (San Diego, CA, USA)) and RNA-Seq (paired-end, 2× 150 bp, 5 GB) on an Illumina Nova-Seq 6000 system to the company Macrogen Europe (Amsterdam, The Netherlands). The raw HTS data were bioinformatically processed and analyzed using GeneiousPrime 2022.2.2 (Biomatters, Auckland, New Zealand) as described in [17]. Sequence similarities between de novo-assembled contigs and sequences of known viruses from the NCBI virus protein and genomic databases were determined by BLASTx and tBLASTx [25]. Contigs with sequence similarity to plant viruses were identified using taxonomic data from NCBI.
The accession numbers of the sequences reported here are PP780273–PP780298 and PP847438–PP847459.

3. Results

3.1. Visual Inspection of Ash Trees Revealed Diversity of Leaf Symptoms

The symptoms we observed on the sampled ash leaves were classified into three categories. Virus-suspected symptoms such as shoestring, chlorotic ringspots, and vein yellowing in combination with leaf deformations were observed both on seedlings and mature seed trees in the seed production site Melzower Forst and on young ashes from Kaisheim (n = 73; Figure 1a,b). Within the canopy of mature seed trees, these symptoms were irregularly distributed and restricted to certain crown areas. In previous studies, they have been linked to an ASaV infection [14]. On young ash trees in Kaisheim, we additionally observed chlorotic line pattern and oak leaf pattern without leaf deformation which, although different from ASaV-typical symptoms, were also classified as virus-suspected (Figure 1c).
Unspecific leaf symptoms such as mottle, chlorotic lesions, chlorotic spots, leaf chloroses, and necrotic lesions were documented at all sampling sites (n = 183; Figure 1d–h), especially in the seed plantations Emmendingen and Schorndorf and the clone archive in Grabenstätt. The proportion of samples showing such symptoms was 85% in Emmendingen, 45% in Schorndorf, and 33% in Grabenstätt.
Leaf material of many trees did not show any symptoms. We collected leaves without symptoms (n = 135) from all sampling sites.

3.2. ASaV Is Associated with Specific Leaf Symptoms

Viruses were detected in samples of all three symptom categories mentioned above. From 391 samples in total, 73 samples (18.7%) displayed virus-suspected symptoms, as seen in Figure 1a–c, while 135 samples (34.5%) showed no symptoms. The majority (183 samples, which corresponds to 46.8%) of all samples displayed symptoms categorized as unspecific, as seen in Figure 1d–h.
From the 73 samples with virus-suspected symptoms, 60 tested positive for ASaV, either as a single infection or mixed infection in combination with PrLBaV or cytorhabdoviruses (Figure 2). These samples all displayed symptoms considered to be ASaV typical, e.g., shoestring, chlorotic ringspots, and vein yellowing in combination with leaf deformations, which is in line with previous studies on the virus [14]. However, not all samples showing ASaV-typical symptoms tested positive, which is discussed later. Disregarding these samples, there is an unambiguous association between ASaV detection and the presence of specific leaf symptoms mentioned above.
Four samples categorized as virus-suspected symptoms displayed line pattern and oak leaf pattern symptoms without leaf deformation (Figure 1c), different from the ASaV-specific symptoms. All of these samples tested positive for mixed infection with PrLBaV and cytorhabdoviruses. Similar symptoms were not observed in single-infected samples for these viruses, indicating that double infections of PrLBaV and cytorhabdovirus are causative for these symptoms. However, this result could not be confirmed for samples from Schorndorf, for which we also detected PrLBaV and cytorhabdovirus double infections in five samples.
The leaf material with unspecific symptoms was shown to be infected with cytorhabdoviruses in most cases. Also, ASaV and PrLBaV single infections were detected in this symptom category. Additionally, mixed infections of all three viruses occurred (Figure 2). In 48% of these samples, no viruses were detected.
We detected viruses in 50% of the samples without symptoms, with cytorhabdoviruses being the most abundant, followed by mixed infections of cytorhabdoviruses and PrLBaV, and PrLBaV single infections (Figure 2).
Together, the abundance of distinct leaf symptoms as described for ASaV points towards an emaravirus infection with a high certainty. However, the absence of virus-suspected symptoms gives no evidence on the virus status of the host, as viruses can infect ash trees in a latent way or induce unspecific symptoms.

3.3. Viruses Are Widely Distributed in Each Stand

The majority of the 391 leaf samples (58.2%) were found to be virus infected.
Cytorhabdoviruses were the most abundant (34.5%) viruses, followed by ASaV (13%), and then PrLBaV, which was detected in 2% of the tested samples. In 8% of the samples, we additionally detected mixed infections, being double infections in most cases. However, triple infections were detected in two ashes in Kaisheim. The PCR results were confirmed by Sanger sequencing of the selected samples. The nepoviruses ArMV and CLRV were not detected by RT-PCR.
Cytorhabdoviruses could be detected at all sites, either as a single infection or mixed infection in combination with ASaV, PrLBaV, or both. ASaV was widely distributed in the natural seed production sites Melzower Forst and Kaisheim. PrLBaV was occasionally detected in the seed plantations Emmendingen and Schorndorf and in Kaisheim. Kaisheim thus showed the highest virus diversity we detected, comprising ASaV, PrLBaV, and cytorhabdoviruses. With the exception of the cytorhabdoviruses, no virus infections were detected in the clone archive of Grabenstätt (Figure 3).

3.4. Sequence Analyses Reveals Distinct Groups of Cytorhabdoviruses

Given the high abundance of cytorhabdoviruses detected in our screening, we aimed to assess their genetic variability. PCR amplicons comprising the partial N-, P-, and L-protein-coding regions were amplified for cytorhabdovirus-infected common ashes from all sampling sites using primers mentioned in Table S1. We identified sequence diversities between the compared sequences at the nucleotide and amino acid level for all genomic regions that were considered.
Referring to the partial NC-protein-coding region located at the 5′ end of the mRNA, sequence comparisons of 557 bp PCR products revealed two groups of sequences. The accession numbers of the sequences are PP780273–PP780298.
The first group included 16 of the 25 analyzed PCR amplicons. These amplicons showed high sequence identities at the nucleotide level (>79%) and the amino acid level (>95%) to each other and to the two amplicons used as references, including a cytorhabdovirus sequence obtained from an infected manna ash (F. ornus) and the recently published cytorhabdovirus sequence (FraGCRV2) from an ash-dieback-diseased common ash ([19], accession number BK064354.1). Samples belonging to this group are colored in blue in Figure S1 (see Supplementary Materials).
The other amplicons showed sequence diversities of more than 25% at the nucleotide and up to 18% at the amino acid level compared to the amplicons of the blue group. However, they displayed high sequence identities (>92% at the nucleotide level and >99% at the amino acid level) to each other, to a cytorhabdovirus sequence obtained from an infected red ash (Fraxinus pennsylvanica, F. penn MRe617), and to a second published cytorhabdovirus sequence (FraGCRV1) from common ash ([19], accession number BK064353.1). The samples that belong to the second group are colored in yellow in Figure S1. Interestingly, one amplicon we generated from an ash located at Melzower Forst (SW24_E62909) displayed high sequence diversity to both groups (Figure S1).
Given the species demarcation criteria established for cytorhabdoviruses by the ICTV, which, among others, include sequence identities of less than 75% for the complete genomes at the nucleotide level and amino acid sequence identities in all cognate open reading frames of less than 80% [17], we assume the two identified groups in our study represent two distinct cytorhabdovirus species affecting Fraxinus as first proposed by [19]. This is supported by the clustering of the sequences into two main phylogenetic groups (Figure 4). One clade comprises the 16 blue-labeled amplicons from Figure S1 and the references from F. ornus and F. excelsior (FraGCRV2). Distinct from this is the second clade comprising the 8 yellow-labeled amplicons from Figure S1, the amplicon for sample SW24_E62909 from Melzower Forst and the reference sequences from F. pennsylvanica and F. excelsior (FraGCRV1). We obtained similar results for the partial P- and L-protein-coding regions. Both cytorhabdovirus species were detected in ashes of all investigated locations (Figure 4).

3.5. Novel Viruses Are Occurring in the Seed Plantations

In 2023, we performed an HTS analyses on leaf and flower material to confirm the results of the RT-PCR-based screening and to search for potentially unknown viruses. Since the proportion of samples showing unspecific symptoms, with a missing connection to identified virus infection, was highest for the seed plantations Emmendingen and Schorndorf, we created two sample pools for each site. The first pool comprised RNA isolated from leaf material from 6 to 10 individual trees. The second pool consisted of RNA isolated from flowers obtained from ten individual trees. A BLASTx search of assembled contigs performed against the NCBI database identified contigs related to cytorhabdoviruses, thereby confirming the wide distribution of these viruses in the seed plantations revealed by the RT-PCR screening (Figure 3). On the other hand, PrLBaV-related sequences could not be retrieved from the pooled samples we investigated by HTS, which might be due to the low abundance of this virus found in the RT-PCR-based survey of the trees in Emmendingen and Schorndorf (Figure 3). Similarly, the sample pool comprising flower material from Emmendingen did not provide sequences related to viruses.
However, we identified contigs related to different plant viruses of the families Betaflexiviridae and Closteroviridae in different sample pools (Table 2). The accession numbers of assembled contigs are PP847438–PP847459.
Contigs with highest similarities to ampelo- and olivaviruses (family Closteroviridae) and a trichovirus (family Betaflexiviridae) were identified in HTS sample pools H0812 and H0813 consisting of leaf material. In the sample pool H0815 consisting of flower material, only sequence contigs related to ampeloviruses (family Closteroviridae) were obtained.
We developed primer pairs from the contigs given in Table 2 for a RT-PCR-based screening of individual leaf and flower samples from the seed plantations. The primer sets were derived targeting the replicase (RdRP), the coat protein (CP), and the heat shock protein (HSP70h) genes due to the HTS contig sequences that mainly covered these regions. Further, these regions are used for taxonomical demarcation criteria [17].
For the novel ampelovirus-like virus, both primer sets (Table S1) generated PCR products of expected sizes for individual samples including leaves and flowers (Figure 5).
The selected PCR products were Sanger-sequenced, thereby confirming their viral origin and the findings of the HTS analyses. In Emmendingen, 35 out of 83 leaf and 3 out of 34 flower samples tested positive for the ampelovirus-like virus by RT-PCR, while in Schorndorf, the putative novel virus was detected in 31 out of 49 leaf and in 13 out of 21 flower samples. The initial screening revealed a wide distribution of the novel ampelo-like virus in the seed plantations. Virus detection so far is attributed to samples showing unspecific or no leaf symptoms, which requires further work to investigate.
For the trichovirus-like virus, the primer set targeting the RdRP (Table S1) yielded a PCR product of the expected size for two individual leaf samples, one from Emmendingen and one from Schorndorf. The PCR products were confirmed by Sanger sequencing. Further investigations on this novel virus and application of the primer sets derived for the olivavirus-like virus detection are still pending.

4. Discussion

Most plant viruses known so far have to be regarded as significant players that impair the health status of deciduous trees and reduce vitality [3]. The number of viruses that have been identified in recent years in several important forest tree species impressively demonstrates their abundance in forest ecosystems [2,26,27]. Considering common ash, which is seriously threatened by ash dieback disease, interdisciplinary studies are meaningful to identify causes of decline and to achieve forestry measures and aid decision making.
In the study presented here, we investigated the virus status of F. excelsior trees in special stands. Special stands were selected due to their relevance, e.g., in delivering high-quality fruits or possessing unique characteristics like tolerance against ash dieback. To ensure vigorous plants and to prevent a spread of viruses in our cultural landscape, it is highly important to address questions about virus infections in such populations.
We detected viruses in the majority of tested samples and at all investigated sites. Specific leaf symptoms can be attributed to an ASaV infection, which is in line with previous studies on other emaraviruses [28,29]. Interestingly, samples showing ASaV-typical symptoms, but which tested negatively, were all taken in October 2021. Due to the upcoming senescense associated with reduced chlorophyll levels and yellowing of leaves, ASaV-induced symptoms were obviously mistakenly addressed as such. Referring the sampling during the summer period, ASaV can be detected with a high degree of reliability by visual observation.
In contrast, no correlation with certain leaf symptoms was found for PrLBaV and cytorhabdoviruses. Symptom expression is influenced by many factors, depending on the virus–host interaction, tree age, and other factors [2]. Although double infections of PrLBaV and cytorhabdoviruses were related to chlorotic line pattern and oak leaf pattern on young ash trees in Kaisheim, this observation could not be confirmed for ash trees in Schorndorf. This might be explained by (a) the overlooking of the symptoms on the Schorndorf trees due to tree height and the irregular symptom distribution within the host, as seen for other viruses [30]; or (b) additional site or host factors that contribute to the symptom expression [2]. Given the irregular distribution of symptoms observed in the crown of mature seed trees from Melzower Forst, a comprehensive inspection of the trees is indispensable to address symptoms properly.
The frequent virus detection in leaf material showing no symptoms and unspecific symptoms, which were considered more likely to result from abiotic factors, emphasizes the need for reliable detection methods to define the virus status of ash trees. Our diagnostic RT-PCR systems targeted the tested viruses with high reliability, as confirmed by sequencing and repeated detection of the viruses in consecutive years for selected trees. The nepoviruses CLRV and ArMV could not be detected by molecular RT-PCR and serological ELISA [31].
In contrast, we identified cytorhabdoviruses as the most abundant viruses in our survey. These viruses often seem to infect ashes with inconspicuous symptoms. Distinct symptoms have been observed for other cytorhabdoviruses, including rice stripe mosaic virus (RSMV). RSMV induces yellow stripes and mosaic on the leaves of infected rice plants, thereby providing a basis for the virus detectability in the field [32]. Particularly, for the seed plantations Emmendingen and Schorndorf, the presence of the cytorhabdoviruses was obvious and widespread with a randomly distributed pattern. Modes of transmission, e.g., vector transmission by insects such as aphids, grasshoppers, and whiteflies, as demonstrated for many cytorhabdoviruses [33] or mechanical transmission remain to be elucidated for the novel cytorhabdoviruses.
Two genetic variants of cytorhabdoviruses were found to occur in ash populations of the investigated sites. Sequence comparisons and phylogenetic analyses of taxonomically relevant gene products revealed sequence diversities and two main clusters of cytorhabdoviruses. Given the species demarcation criteria for cytorhabdoviruses, which include sequence identities of less than 75% for the complete genomes at the nucleotide level and amino acid sequence identities in all cognate open-reading frames of less than 80% [17], these genetic variants could represent independent species, supported by the findings of [19]. Ongoing genetic characterization of the whole genome will shed more light on this. We identified the blue and yellow variants not only in F. excelsior but also in F. ornus and F. pennsylvanica, which is, to our best knowledge, the first detection of these viruses in red and manna ash. Strikingly, one of the viruses (FraGCRV2) identified by [19] was associated with H. fraxineus., the causative agent of ash dieback in common ash. The consequences of this for the viral life cycle and transmission processes remains to be studied in detail. However, it highlights the possible virus–fungi interaction and the demand for further investigations on the cytorhabdovirus epidemiology, diversity, and biology in ash.
Thanks to modern sequencing technologies, the understanding of plant viromes has been greatly improved, including the detection of new viruses without a priori knowledge [1]. Since unspecific leaf symptoms, indicative of putative viral origin, were most frequently observed in Emmendingen and Schorndorf, we applied HTS on mixed sample pools of these seed plantations. On the one hand, this confirmed the occurrence of cytorhabdoviruses in Emmendingen and Schorndorf seen through the RT-PCR. Additionally, we identified novel viruses of the Betaflexiviridae and Closteroviridae families in the mixed sample pools. In contrast to the Closteroviridae contigs, sequence contigs related to the Betaflexiviridae family were only identified in sample pools consisting of leaf material but not in the pool that contained flowers. Whether this is due to the virus accumulation being restricted to infected leaves, a general low abundance of the virus within the ash populations, or due to the sampling procedure of the flowers, which were selected by chance and did not rely on symptom expression, cannot be conclusively stated at this point. Testing of leaves and flowers from individual trees will provide clarification on this. Primers sets that target the novel-identified viruses were derived from genomic regions used for genetic investigations in previous studies, making them promising candidates for the RT-PCR screening [34,35,36]. A wide distribution of the ampelovirus-like virus was determined within the plantations of Emmendingen and Schorndorf. Extending the testing to further sites and trees is necessary to explore their distribution within ash populations and estimate their significance for the ash health. The results achieved so far indicate that novel viruses must be given attention. Viruses already known for a long time to affect common ash including CLRV and ArMV [10], were not detected in this study, while recently detected viruses, including ASaV, PrLBaV, and cytorhabdoviruses, are widely distributed in the five ash stands. Furthermore, undiscovered viruses that contribute to the ash virome cannot be excluded at present.
Since all sampling sites were shown to contain virus-infected ashes, more focus should be placed on viruses, their characteristics and their interactions with other biotic factors including H. fraxineus. Only under these conditions, a comprehensive view on ash dieback and strategies to overcome the disease can be provided.
Together, our results provide valuable knowledge on viruses in F. excelsior, which is mandatory to deepen the view on the factors involved in the health status of ashes and help to preserve them as an economically important tree species.

5. Conclusions

Plant viruses are widely distributed in all investigated special stands of common ash, with single and mixed infections of viruses belonging to different families being found to occur. In the natural seed production stands, Melzower Forst and Kaisheim, the emaravirus ASaV is widely distributed. We documented a close association between the pathogen detection and the specific disease symptoms on leaves, such as shoestring, mottle, and chlorotic ringspots, in combination with deformation, which can clearly be distinguished from symptoms caused by other pathogens. Cytorhabdoviruses were detected at all sampling sites, comprising two main groups with considerable sequence diversities. The idaeovirus PrLBaV occurs in the seed plantations Emmendingen and Schorndorf and at Kaisheim. Cytorhabdoviruses and PrLBaV cannot be linked to macroscopically visible symptoms, and their impact on the tree physiology is still unknown. Novel viruses of the Betaflexiviridae and Closteroviridae families have been identified both by HTS in mixed sample pools and by RT-PCR in individual trees. The frequent virus detection in samples without virus-suspected symptoms, including those without symptoms at all, requires a consequent testing to assess the virus status accurately. More intensive studies worldwide are needed to clarify the role of viruses in the tree and microbiome system. Their connections to ash dieback disease, however, remains to be elucidated in the future.

Supplementary Materials

The following supporting information are available online at https://www.mdpi.com/article/10.3390/f15081379/s1, Table S1: Primer pairs used for RT-PCR-based virus detection [14,16,37,38]; Figure S1: Nucleotide sequence identity matrix (lower diagonal) and amino acid sequence identity matrix (upper diagonal) of partial cytorhabdovirus PCR amplicons of the N-terminal part encoding the nucleocapsid protein.

Author Contributions

Conceptualization, M.R.; methodology, M.R., K.K. and R.A.K.; software, M.R. and K.K.; validation, R.A.K., K.K., S.v.B. and C.B.; formal analyses, M.R.; investigation, M.R., K.K. and R.A.K.; resources, S.v.B. and C.B.; data curation, M.R.; writing—original draft preparation, M.R.; writing—review and editing, K.K., R.A.K., S.v.B. and C.B.; visualization, M.R.; supervision, S.v.B.; project administration, C.B.; funding acquisition, C.B. All authors have read and agreed to the published version of the manuscript.

Funding

This project receives funding via the Waldklimafonds (WKF) funded by the German Federal Ministry of Food and Agriculture (BMEL) and Federal Ministry of Environment, Nature Conservation, Nuclear Safety and Consumer Protection (BMUV) administrated by the Fachagentur Nachwachsende Rohstoffe (FNR) under grant agreement No.: 2220WK40B4.

Data Availability Statement

The HTS raw reads used in the presented study are openly available in the Sequence Read Archive (SRA) database (NCBI) under accession number PRJNA1113958.

Acknowledgments

We acknowledge Stefanie Wohlfahrt and Hector Fernandez Colino for technical support in the preparation of ash leaf material for virus detection and Lisa Buchner from the department Physical Geography/Landscape Ecology and Sustainable Ecosystem Development, Catholic University Eichstätt-Ingolstadt (KU) for providing leaf material from mature seed trees from the seed production site Kaisheim. We thank Thomas Gaskin for English proofreading of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design, execution, interpretation, or writing of the study.

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Forests 15 01379 g001
Figure 1. Leaf symptoms on common ash (F. excelsior) sampled at different locations within the study. Virus-suspected symptoms were observed including (a) chlorotic ringspots and leaf deformation on a seedling from Melzower Forst, (b) shoestring on a mature seed tree from Melzower Forst, and (c) chlorotic line pattern and oak leaf pattern without deformation on a young ash tree in Kaisheim. Unspecific symptoms were observed, including (d) mottle on a seedling from Melzower Forst, (e) chlorotic lesions and (f) chlorotic spots on ash trees from the seed plantation Emmendingen, (g) unspecific leaf chlorosis on an ash tree from the seed plantation Schorndorf, and (h) necrotic lesions on an ash tree from the clone archive in Grabenstätt.
Figure 1. Leaf symptoms on common ash (F. excelsior) sampled at different locations within the study. Virus-suspected symptoms were observed including (a) chlorotic ringspots and leaf deformation on a seedling from Melzower Forst, (b) shoestring on a mature seed tree from Melzower Forst, and (c) chlorotic line pattern and oak leaf pattern without deformation on a young ash tree in Kaisheim. Unspecific symptoms were observed, including (d) mottle on a seedling from Melzower Forst, (e) chlorotic lesions and (f) chlorotic spots on ash trees from the seed plantation Emmendingen, (g) unspecific leaf chlorosis on an ash tree from the seed plantation Schorndorf, and (h) necrotic lesions on an ash tree from the clone archive in Grabenstätt.
Forests 15 01379 g001
Forests 15 01379 g002
Figure 2. Association between leaf symptoms and virus detection. The number of samples for each category is given in brackets. Viruses were detected in leaf material, regardless of the occurrence of symptoms. While ASaV detection was closely associated with symptoms including shoestring, chlorotic ringspots and vein yellowing, infections with PrLBaV and cytorhabdoviruses could not be linked to specific symptoms.
Figure 2. Association between leaf symptoms and virus detection. The number of samples for each category is given in brackets. Viruses were detected in leaf material, regardless of the occurrence of symptoms. While ASaV detection was closely associated with symptoms including shoestring, chlorotic ringspots and vein yellowing, infections with PrLBaV and cytorhabdoviruses could not be linked to specific symptoms.
Forests 15 01379 g002
Forests 15 01379 g003
Figure 3. Results of the virus screening for all sampling sites are given as pie charts with each sector representing one virus and its proportion to the overall virus diversity that was detected. N indicates the number of samples tested for each site. Viruses are indicated in different colors, as given in the legend.
Figure 3. Results of the virus screening for all sampling sites are given as pie charts with each sector representing one virus and its proportion to the overall virus diversity that was detected. N indicates the number of samples tested for each site. Viruses are indicated in different colors, as given in the legend.
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Forests 15 01379 g004
Figure 4. Cytorhabdoviral sequences cluster into two main groups. Amino acid sequences comprising the partial N protein were aligned using MUSCLE, and phylogenetic trees were built using neighbor-joining methodology in Geneious prime software 2022.2.2. Branches report bootstrap support (1000 replicates). Potato yellow dwarf virus (GU734660, Alphanucleorhabdovirus) was used as an outgroup. The two clades are colored in blue and yellow, in line with the sequence identity matrix at nucleotide and amino acid level for all amplicons given in Supplemental Figure S1. The location of each sample is given on the right. EM—Emmendingen, MF—Melzower Forst, KH—Kaisheim, SCH—Schorndorf, GS—Grabenstätt.
Figure 4. Cytorhabdoviral sequences cluster into two main groups. Amino acid sequences comprising the partial N protein were aligned using MUSCLE, and phylogenetic trees were built using neighbor-joining methodology in Geneious prime software 2022.2.2. Branches report bootstrap support (1000 replicates). Potato yellow dwarf virus (GU734660, Alphanucleorhabdovirus) was used as an outgroup. The two clades are colored in blue and yellow, in line with the sequence identity matrix at nucleotide and amino acid level for all amplicons given in Supplemental Figure S1. The location of each sample is given on the right. EM—Emmendingen, MF—Melzower Forst, KH—Kaisheim, SCH—Schorndorf, GS—Grabenstätt.
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Forests 15 01379 g005
Figure 5. Detection of the novel ampelovirus-like virus. Gel electrophoresis of the fragments amplified by RT-PCR for three samples, applying two sets of primer targeting the RdRP- and the CP-coding regions of the virus. Lanes 2 and 6, 3 and 7, and 4 and 8 represent the same samples, respectively. Lanes 5 and 9 represent the water controls for each primer set. Lane 1 represents the Gene Ruler 1 kb DNA Ladder (Thermo Scientific). The genomic regions targeted by the two primer sets are given on the right.
Figure 5. Detection of the novel ampelovirus-like virus. Gel electrophoresis of the fragments amplified by RT-PCR for three samples, applying two sets of primer targeting the RdRP- and the CP-coding regions of the virus. Lanes 2 and 6, 3 and 7, and 4 and 8 represent the same samples, respectively. Lanes 5 and 9 represent the water controls for each primer set. Lane 1 represents the Gene Ruler 1 kb DNA Ladder (Thermo Scientific). The genomic regions targeted by the two primer sets are given on the right.
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Table 1. Sampling sites investigated in the study; N: North, E: East.
Table 1. Sampling sites investigated in the study; N: North, E: East.
StandFederal
State		Location TypeGeographical Location
EmmendingenBaden-Württembergseed
plantation		48°6′38.50″ N, 7°52′20.49″ E
SchorndorfBaden-Württembergseed
plantation		48°46′35.59″ N, 9°25′31.00″ E
KaisheimBavariaseed
production site		48°48′20.83″ N, 10°47′33.34″ E
GrabenstättBavariaclone
archive		47°50′28.62″ N, 12°30′41.87″ E
Melzower ForstBrandenburgseed
production site		53°11′10.86″ N, 13°57′14.30″ E
Table 2. Assembled contigs related to novel viruses identified in ash sample pools by HTS in 2023, rather than the ones detected in RT-PCR. Query coverage, E-value and sequence identity are given in comparison with the best match in BLASTx.
Table 2. Assembled contigs related to novel viruses identified in ash sample pools by HTS in 2023, rather than the ones detected in RT-PCR. Query coverage, E-value and sequence identity are given in comparison with the best match in BLASTx.
Sample PoolContig NumberSize
(nt)		Best Match in BLASTx (Accession Number, Virus Genus/Family)Query Coverage
(%)		E-ValueSequence Identity
(%)
H0812 Emmendingen leafC70432473GKSV * (QDC33513.1, trichovirus/Betaflexiviridae)696 × 10−1146
C76842352GKSV (QCZ35721.1, trichovirus/Betaflexiviridae)292 × 10−9264
C114441809GKSV (QTF33920.1, trichovirus/Betaflexiviridae)995 × 10−17250
C11345370GLRaV-1 *1 (AHB87103.1, ampelovirus/Closteroviridae)215 × 10−4547
C26583981GLRaV-1 (QBZ78745.1, ampelovirus/Closteroviridae)380.056
C63572625GLRaV-1 (AXL94955.1, ampelovirus/Closteroviridae)6410−15845
C107071894GLRaV (ADJ95800.1, ampelovirus/Closteroviridae)313 × 10−7284
C27877727OLYaV *2 (UXN85457.1, olivavirus/Closteroviridae) 894 × 10−8757
H0813 Schorndorf leaf **C39772987ACLSV *3 (UCJ00976.1, trichovirus/Betaflexiviridae)956 × 10−10433
C64462309GKSV (QDC33518.1, trichovirus/Betaflexiviridae)640.060
C93061838GKSV (QEV82110.1, trichovirus/Betaflexiviridae)993 × 10−15446
C16410733PAVA *4 (YP_010086802.1, ampelovirus/Closteroviridae)1410−14349
C5407259PAVA (YP_010086802.1, ampelovirus/Closteroviridae)1710−10146
C7796068GLRaV-1 (AHB87153.1, ampelovirus/Closteroviridae) 2110−4940
C90121878OLYaV (QOK36435.1, olivavirus/Closteroviridae) 782 × 10−14347
C21850830OLYaV (UXN85457.1, olivavirus/Closteroviridae)2710−3077
C28227594OLYaV (QOK36430.1, olivavirus/Closteroviridae)992 × 10−7867
C31337524OLYaV (UXN85452.1, olivavirus/Closteroviridae)9910−8382
H0815 Schorndorf flowerC1595434PAVA (YP_010086802.1, ampelovirus/Closteroviridae)289 × 10−14449
C4993343GLRaV-13 (BDX29264.1, ampelovirus/Closteroviridae)64 × 10−1751
C21341878GLRaV-1 (ARP51762.1, ampelovirus/Closteroviridae)4510−10662
C51901191GLRaV-3 (AOS89854.1, ampelovirus/Closteroviridae)984 × 10−8146
* Grapevine Kizil Sapak virus; *1 Grapevine leafroll-associated virus 1; *2 Olive leaf-yellowing-associated virus; *3 Apple chlorotic leaf spot virus; *4 Pistachio ampelovirus A; ** for H0813 only the three longest contigs relating to viruses of the Betaflexiviridae and Closteroviridae families are given.
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Rehanek, M.; Al Kubrusli, R.; Köpke, K.; von Bargen, S.; Büttner, C. Detection of Viruses in Special Stands of Common Ash Reveals Insights into the Virome of Fraxinus excelsior. Forests 2024, 15, 1379. https://doi.org/10.3390/f15081379

AMA Style

Rehanek M, Al Kubrusli R, Köpke K, von Bargen S, Büttner C. Detection of Viruses in Special Stands of Common Ash Reveals Insights into the Virome of Fraxinus excelsior. Forests. 2024; 15(8):1379. https://doi.org/10.3390/f15081379

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Rehanek, Marius, Rim Al Kubrusli, Kira Köpke, Susanne von Bargen, and Carmen Büttner. 2024. "Detection of Viruses in Special Stands of Common Ash Reveals Insights into the Virome of Fraxinus excelsior" Forests 15, no. 8: 1379. https://doi.org/10.3390/f15081379

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