Identification Of Virus For Oak Health

Health status of oak trees in a conservation seed orchard has been observed for over twenty years, focusing on characteristic virus-suspected symptoms. Screen the oak seedlings in three consecutive years, use a newly established virus-specific diagnostic reverse transcription polymerase chain reaction (RT-PCR) and find out the causes and impact of oak decline so that it would be possible to reforest high-quality seedlings of oak successfully in a time of climate change. By Martina Bandte, Marius Rehanek, Susanne von Bargen and Carmen Büttner, Albrecht Daniel Thaer-Institute for Crop and Animal Sciences, Bertram Leder, Forestry and Timber North Rhine-Westphalia

The health status of oak trees in a conservation seed orchard has been observed for over twenty years, focusing on characteristic virus-suspected symptoms. The orchard was established in 1992 in Kreuztal, North Rhine-Westphalia (Germany) with 1302 seedlings in 186 clusters. 

The number of seedlings showing chlorotic ringspots and mottle on leaves has fluctuated annually, but has increased from 3.3 percent to 12.1 percent in the last 20 years; the number of affected clusters has risen from 8 percent to 25.9 percent. A scientific breakthrough was the identification of a novel virus related to members of the genus Emaravirus in diseased oak by high-throughput sequencing (HTS). 

Screening of the oak seedlings in three consecutive years, using a newly established virus-specific diagnostic reverse transcription polymerase chain reaction (RT-PCR), confirmed the virus infection and revealed a close to 100 percent association between the observed leaf symptoms and the novel virus. 

As no other plant virus could be identified in the HTS-datasets, we assume the novel virus is primarily causing the symptoms. To reliably detect the novel virus in oaks, RT-PCR targeting the viral RNA3 or RNA4 should be applied in routine testing of symptomatic leaf tissue.


Widespread Species On Decline

Quercus robur (L.), commonly known as common oak, pedunculate oak, or European oak, is a very widespread species which is native to most of Europe and described as a vigorous tree with a large ecological amplitude. 

Oaks are amongst the most economically and ecologically important deciduous trees in Europe providing wood for fuel, bark for tanning, timber for construction, and acorns for livestock. Across 34 European countries, pedunculate oak covers approximately 49,000 sq km. 

In Germany, oak species can be found on around 10 percent of forested land, making oaks the second most important deciduous tree species after European beech (Fagus sylvatica L.). The production of high-quality wood is associated with long rotations and high labour costs.

Dieback and decline in oak have been reported in Europe since the early 1900s as well as in the most recent decades. On the basis of historical records and dendrochronological measurements, oak decline in Central Europe has been attributed to single or combined effects of various abiotic factors such as air pollution, nitrogen eutrophication, soil chemical stress and climatic extremes (winter frost, summer drought), defoliating insects, and pathogenic fungi. However, little attention has been paid to plant viruses, despite classifying this group of organisms as a presumed contributing factor in tree decline as early as 1985. 

Plant viruses are rumoured to lead to early senescence of trees, which is known to reduce the regeneration capacity of the host plants, and the juvenile metabolic vigoro is lost prematurely. Thereby, virus-infected trees have a reduced potential for recovery from omnipresent abiotic stress conditions compared to non-infected trees.

Most plant diseases have adverse effects on plant growth and productivity, which can range from relatively low tree mortality rates to 100 percent losses for certain plant species. Co-infection with multiple viruses may enhance pathogenicity. 

Although the impact of plant viruses on forest trees remains to be investigated, limited information concerning fruit trees is available. For instance, it showed that yield efficiencies of peach infected with a mild isolate of plum pox virus (PPV) did not differ statistically although trees produced slightly more fruit of smaller size that ripened earlier than non-infected trees. 

Mixed infections of PPV, prune dwarf virus (PDV), and prunus necrotic ringspot virus (PNRSV) have been shown to reduce growth and to exhibit bark canker, trunk malformation, and tree mortality in some peach cultivars. The synergistic effect of PPV with other viruses resulted in a growth reduction of the seedlings by 2.9 to 69.1 percent.

Surveys in North German nurseries and of several German forest districts on the health status of European oak (Quercus robur L.) led to the observation of many seedlings and trees with characteristic virus-like symptoms such as chlorotic ringspots, chlorotic spots, and mottling. 

Some of these plants had degenerated twigs and suffered from a distinct loss of vigoro. Already in the 1970s, these symptoms had been described and following visual inspection assumed to be induced by a viral pathogen. 

However, neither the etiology nor the epidemiology of a viral agent has so far been described. In order to demonstrate the biotic nature of the virus-like symptoms and to identify the putative agent, we initially focused on symptomatic trees. Virus-like symptoms on European oak as well as therewith confusable discolorations are described by. 

In 1996, carried out experiments testing graft-transmission of the assumed agent and stated that the symptoms are induced by an infectious agent. Transmission to herbaceous plants by mechanical inoculation failed in these and later experiments. 

The authors ruled out the presence of tobacco mosaic virus (TMV), tobacco necrosis virus (TNV), cherry leaf roll virus (CLRV), and brome mosaic virus (BMV), which are known to occur not only in forest ecosystems but are also associated with Quercus sp.. 

Recent advances in high-throughput sequencing (HTS) technologies and bioinformatics have generated unique opportunities for discovering and diagnosing plant viruses and viroids. Thus, during the last decade the application of this technology has led to the discovery of more than one hundred new plant viruses, new virus variants, or new plant hosts of known viruses, which has shed light on the complex world of microbial communities in environmental samples.

Advances in knowledge on the causes and impact of oak decline have been slow, being partly due to the complex nature of the problem. Therefore, we aimed to (i) observe the health status of oak trees in a conservation seed orchard over a period of more than twenty years by visual means, (ii) document the characteristic virus-suspected symptoms, (iii) identify a novel virus by HTS in diseased oak, and (iv) verify the association of the novel virus with the chlorotic ringspots via the development and application of a virus-specific reverse transcription-polymerase chain reaction (RT-PCR).


Conservation Seed Orchard

The selected orchard with common oak was established in 1992 in Kreuztal, North Rhine-Westphalia (Germany) with three-year-old seedlings (origin 817 01, provenience ‘’von Plettenberg/Hovestadt’’). The 3.1 ha oak cluster planting established on former farmland comprises 186 groups consisting of seven seedlings each. 

These seedlings were planted with an initial spacing of 1 m and at a distance (centre to centre) of 12 m between the groups. The site is 281 m above sea level. The long-term mean annual rainfall is 859 mm, and the mean annual temperature is 8.5 deg C.

Since 1995, the orchard was inspected at least once a year, focusing on the characteristic virus suspected leaf symptoms. Inspection and sampling took place in late spring/early summer. During this period leaf symptoms were mostly very clearly visible and not yet masked by other biotic or abiotic factors. Symptomatic leaf samples were always taken from individual trees. For comparison, we included leaf material from trees not exhibiting virus-suspected symptoms.


RNA-sequencing & Analysis

In 2014, new molecular tools enabled further steps to unravel the causal agent inducing the disease. Leaf material from a single diseased common oak (E53309, Quercus robur L.) tree from the seed conservation orchard was sampled. 

The leaves of this tree had shown characteristic chlorotic ringspots and mottling over several years. For RNA-sequencing (RNASeq) (Illumina, HiSeq), total RNA was isolated from 50 mg of tissue showing chlorosis and necrosis, excised from leaves (Invitrap Spin Plant RNA Mini Kit, Invitek Molecular, Berlin, Germany), followed by mRNA enrichment (Dynabeads mRNA purification Kit, Thermo Fisher Scientific, Hennigsdorf, Germany), purification, and DNAse digest. 

Single-read sequencing was performed with Illumina’s sequencing-by-synthesis approach using an RNA sample preparation kit, a single-read cluster generation kit v3, and the TruSeq mRNA sample prep kit v3 (Illumina Inc., San Diego, CA, US). 

Sequencing of barcoded libraries was performed on an Illumina HiSeq 2500 system (Illumina Inc., San Diego, CA, USA). De novo assembly of reads of this sample was performed by selecting standard parameters and using a CLC Genomics Workbench V7.0.1 (Qiagen, Aarhus, Denmark). 

Taxonomic binning was performed using MEGAN (MEtaGenome Analyzer, version X, University Tübingen, Germany) and by applying one read support parameter, while the Basic Local Alignment Search Tool X (BLASTX, National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) comparison against the nr protein database (NRPROT) of assembled contigs with a minimal size of 300 nucleotides was run under default parameters for identifying nucleotide entries for viruses in GenBank (NCBI). 

In 2016, a high-throughput sequencing (RNASeq, Illumina Inc., San Diego, CA, USA) of a diseased tree from the same seed orchard was performed for emaraviral sequences amplified by use of the generic PDAP213 primer. 

RNA-extraction from a pooled sample of leaf material collected from a diseased tree in June 2016 (E54889) showing chlorotic ringspots and mottle and ds-cDNA preparation for library generation for RNASeq carried out by the Company BaseClear B.V. (Leiden, Netherlands); data analyses, including assembly of contigs, were then conducted as described in.


RT-PCR For Plant Virus Diagnosis

Starting in 2016, we conducted RT-PCR analysis to both confirm viral sequences identified by HTS in diseased oaks and to study the association of the virus with observed symptoms. To this end, leaf material from the seed conservation orchard was sampled and was immediately used for isolation of total RNA or stored at −20 deg C beforehand.

Total RNA was isolated according to and reverse transcribed into cDNA by the use of random hexamer primer and Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific Inc., Waltham, MA, USA) following manufacturer’s instructions. 

Synthesised cDNA was used as template for conventional RT-PCR. The mitochondrial expressed nad5 gene was verified according to as an internal control. RT-PCR-based detection of the different viral genome components applied the genus-specific primer pair motif A sense and motif C antisense as well as HTS-derived species-specific primers. 

PCR amplification was conducted in a thermal cycler (BioRad Laboratories GmbH, Feldkirchen, Germany) with an initial denaturation at 94 deg C for 3 min, followed by cyclic amplification for 35 cycles at 94 deg C for 30 s, at 50 deg C (RNA1 and RNA2)/54 deg C (RNA3)/55 deg C (RNA4) for 30 s, and at 72 deg C for 30 s. 

The PCR products were separated by 1–1.5 percent-agarose gel electrophoresis. Amplified PCR products were subsequently sequenced by Sanger Sequencing in both directions using PCR product specific primers.


Relationship Between Virus & Performance

Out of 186 groups planted in 1992, only 162 remain to date. Within 27 years of the orchard being established, the total number of trees has decreased from 1302 to 422. A natural selection of ideally one tree per group is desired and was reached by 44 (data not shown). 

The number of seedlings showing the characteristic virus associated symptoms is subject to large fluctuations. For example, in 2013, only 17 trees were classified as virus-infected, but 43 and 51 were classified thusly in 2008 and 2018, respectively. 

During the last 20 years, the number of affected groups increased significantly from 14/175 (8 percent) to 42/162 (25.9 percent). During this period, the number of individual trees with characteristic symptoms increased from 19/577 (3.3 percent) to 51/422 (12.1 percent). In 2016, a molecular diagnostic system was set up and enabled the detection of the novel agent causing these symptoms in following years.

In recent years there have been fluctuations in the number of diseased single trees with visible characteristic leaf symptoms. While 2015 to 2017 show a relatively constant occurrence of emaravirus-infected trees, 2014 and 2018 stand out, with the number of diseased tress being clearly below and above the average, respectively. This phenomenon applies to both infected individual trees and the number of affected groups.

The number of established oak groups in 1992 was anticipated as also being the future number of crop trees. A first thinning, to remove all other remaining oaks in the group to provide growing space for the single prevailing crop tree, has yet to be applied. To date, 44 of the remaining 162 groups consist of one prevailing crop oak. Of these oaks, seven (15.9 percent) were infected with the novel emaravirus. 

In another 43 groups, neither of the last two remaining trees were able to prevail. Remarkably, in nine of those groups, one oak tree was infected with the virus, in four groups both were infected. A total of 64 groups consist of one or two healthy oak trees. 

These groups are distributed throughout the site and cluster; only three single crop trees stand individually. The 24 groups being lost, without a viable prevailing oak, cluster in a northeast and southwest orientation. In four of these groups, trees with characteristic virus-associated symptoms were observed in previous years. 

When analysing the distribution of the 42 groups affected by the virus, it was obvious that most of these groups cluster. Of these, only five are offside and do not border on affected groups of plants. Interestingly, only at the centre of the site, an area of about 20 groups consists of symptomless seedlings. We did not detect any virus symptoms within the entire observation period in these groups.


Mild Strain Cross-protection

Through the survey as above, we carried out a comprehensive screening of oak trees in the seed orchard and confirmed (i) the suitability of RT-PCR targeting the viral RNA3 and RNA4 for routine testing and (ii) a clear association between the detection of viral genome segments and chlorotic ringspot on the leaves of diseased trees. 

Since HTS datasets gave no hints to the involvement of other plant viruses, we assume that the novel virus is the primary cause of the observed symptoms. As the genetic composition of emaraviruses is variable with respect to genome segments, we cannot rule out the possibility that the novel virus may have other genome segments. Further molecular characterisation is essential. The illumination of biological features is also required.

Although not all emaraviruses have been biologically characterised, various modes of transmission have been proven, including eriophyid mites, grafting, mechanical means (inoculation and contaminated cutting tools), and (rarely) seeds. 

In the current study the proportion of virus-infected trees increased from three to 12 percent during the observation period. The distribution of these trees suggests a vector transmission of the virus, as already shown for some other viruses from the genus emaravirus. 

However, so far neither a vector nor an artificial mechanical transmission has been confirmed. Furthermore, it remains unclear why the virus is not inevitably more often transmitted to adjacent seedlings within the same cluster, rather than to neighbouring cluster. In this context, virus resistance has to be considered, with future investigations exploring plant defence.

Both biotic and abiotic conditions shape plant responses to stress events. The environment is rarely ideal for plant growth and even mild; episodic stresses can predispose plants to inoculum levels they would otherwise resist. 

Most plant viruses are described as pathogens causing diseases in agricultural and horticultural crops. They are harmful to their host as they are dependent on host resources to support their own reproduction and dissemination. 

It is becoming evident that the nature of the virus–host relationship is dependent on the environment. For example, a virus being pathogenic under normal environmental conditions can become beneficial to the host under stressful conditions.

This is true when the virus can ameliorate the impacts of biotic stress and can help plants to combat abiotic stress. White clover plants, for example, are less attractive to fungal gnats when they are infected with White clover mosaic virus, and wild gourds are less attractive to beetles when they are infected with Zucchini yellow mosaic virus. 

The capacity of a mild strain of a virus can also be used to protect the plant against subsequent infection by a severe strain of the virus. This is known as mild strain cross-protection and has proven to be a powerful approach in combating devastating pathogens such as Citrus tristeza virus and Pepino mosaic virus. 

Infection with plant viruses, independent of the width of their host range, can actually provide their host protection from drought stress. In virus-infected tobacco, beet, and rice, drought symptoms appeared later and leaves were able to maintain water longer than uninfected counterparts. 

Barley yellow dwarf virus-infected wheat was also associated with higher leaf water potential when water inputs were low and infected plants surpassed control plants in performance traits such as above-ground growth, seed set, seed yields, and seed germination. 

The physiological mechanisms responsible for virus-conditioned resistance to drought stress still remain to be elucidated. It was suggested that the protection may be due to a virus-induced accumulation of osmoprotectants and antioxidants such as anthocyanins. 

Likewise, proline, a proteinogenic amino acid acting on osmotic adjustment that helps subcellular structure stabilisation and the elimination of free radicals, invariably had a higher concentration in sweet orange (Citrus sinensis) infected with citrus tristeza virus. 

Survival of infected plants under extreme drought stress represents a conditional difference in fitness, assuming the surviving virus-infected plants can subsequently produce offspring. 

It is essential to investigate the influence of viruses on tree fitness and populations in environmental contexts further. However, demonstrating that a virus benefits a plant host is difficult as in an ecosystem the virus and host do not exist in isolation. The benefit or harm of a virus for the plant probably depends therefore on how interactions between the virus and other players in the community affect their composition and, ultimately, the fitness of the host.

Although the association between the observed leaf symptoms and the novel emaravirus in the oak conservation seed orchard is close to 100 percent, the association between the detection of the virus and the overall performance of the seedlings is non-existent or at best weak. Thus, a virus-infected seedling may even emerge to a clearly prevailing tree. Reliable data are currently lacking for forecasts on the growth behaviour of virus-infected oak trees in the future.

Besides, oak seed orchards support the conservation as well as the development of forest genetic resources. High-quality seedlings are necessary for successful reforestation in a time of climate change. Quality traits must be considered in particular if a seedling has to have a high survival and growth capacity after transplanting in an unfavourable environment. 

Unfortunately, it is not possible to ascribe a universal set of structural or physiological high-quality attributes to plants. Traits of seedlings conferring high performance on one site do not necessarily maximise seedling outplanting performance on another.

Thus, it is conceivable that plant viruses may extend survival of their hosts under conditions of abiotic stress that could benefit hosts if they can subsequently recover and reproduce. Such beneficial effects of viruses have been poorly studied so far. Perhaps we need to break completely new ground to enhance silviculture.

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