Influence of Mechanical Wounding and Compartmentalization Mechanism on the Suppression of Invasive Plant Species Using the Example of Cherry Laurel (Prunus laurocerasus)

Natural habitats increasingly face the introduction and spread of non-native species. Under the right conditions, non-native species can become invasive over time. This issue is now being addressed by many experts and researchers who are using and developing various approaches and methods to limit and eliminate or suppress problematic plant species. Many invasive plants are already spreading uncontrollably in urban and forestry areas, causing health hazards, environmental and economic damage and negatively impacting natural ecosystems. The use of chemical agents is generally limited, so our only option to control and suppress the problematic species is mechanical removal. In this research suppression by tree stem wounding, i.e., incomplete girdling, was used. This type of injury causes the plant to lose its vitality, become weaker after first year and then die within a few years. Using a research approach, we chronologically monitored the response of cherry laurel (Prunus laurocerasus L.) stem tissue to mechanical wounding of the incomplete girdling. Magnetic resonance imaging (MRI) and light microscopy were used for monitoring moisture content and anatomical changes in different periods after injury. The results of the study showed that cherry laurel, with an intense wound tissue response and other changes, is a species with good compartmentalization potential. The rapid and intense tissue response to injury requires high energy and nutrient consumption and consequently leads to a loss of vigour and mechanical stability, which may result in plant destruction. Results revealed that mechanical wounding by incomplete girdling is a successful method for suppression of non-native and invasive cherry laurel.

Identification and Molecular Analysis of Putative Self-Incompatibility Ribonuclease Alleles in an Extreme Polyploid Species, Prunus laurocerasus L.

Cherry laurel (Prunus laurocerasus L.) is an extreme polyploid (2n = 22x) species of the Rosaceae family where gametophytic self-incompatibility (GSI) prevents inbreeding. This study was carried out to identify the S-ribonuclease alleles (S-RNases) of P. laurocerasus using PCR amplification of the first and second intron region of the S-RNase gene, cloning and sequencing. A total of 23 putative S-RNase alleles (S1–S20, S5m, S13m, and S18m) were sequenced from the second (C2) to the fifth conserved region (C5), and they shared significant homology to other Prunus S-RNases. The length of the sequenced amplicons ranged from 505 to 1,544 bp, and similar sizes prevented the proper discrimination of some alleles based on PCR analysis. We have found three putatively non-functional alleles (S5m, S18m, and S9) coding for truncated proteins. Although firm conclusions cannot be drawn, our data seem to support that heteroallelic pollen cannot induce self-compatibility in this polyploid Prunus species. The identities in the deduced amino acid sequences between the P. laurocerasus and other Prunus S-RNases ranged between 44 and 100%, without a discontinuity gap separating the identity percentages of trans-specific and more distantly related alleles. The phylogenetic position, the identities in nucleotide sequences of the second intron and in deduced amino acid sequences found one or more trans-specific alleles for all but S10, S14, S18, and S20 cherry laurel RNases. The analysis of mutational frequencies in trans-specific allele pairs indicated the region RC4–C5 accepts the most amino acid replacements and hence it may contribute to allele-specificity. Our results form the basis of future studies to confirm the existence and function of the GSI system in this extreme polyploid species and the alleles identified will be also useful for phylogenetic studies of Prunus S-RNases as the number of S-RNase sequences was limited in the Racemose group of Prunus (where P. laurocerasus belongs to).

Invasions of alien woody plant taxa across a cluster of villages neighbouring the Mlyňany Arboretum (SW Slovakia)

Ornamental plantations in cities and particularly botanical gardens and arboreta are rich sources of alien flora. Mlyňany Arboretum, established in 1892, cultivates 1049 non-native woody plant species on the area of 67 ha. In this work we answered following questions: 1. How many taxa are spontaneously spreading in the arboretum and how is the spreading intensity related to their ecological demands and reproduction traits? 2. How many taxa appear behind the fence? 3. How far from the arboretum they can get? 4. Do private gardens and historical aristocratic park in the studied village cluster contribute to species escapes from culture? 5. Which from the widely spread taxa can represent future risk of invasiveness on the national level? We found that about one tenth of taxa spread across the arboretum (particularly Cotoneaster spp., Prunus laurocerasus, P. serotina and Quercus rubra) and number of their seedlings corresponded only with the mother plant number. Almost one third of these species left the arboretum and their seedlings were observed in distance up to 500 m from the village (mainly Mahonia aquifolium, P. serotina). Private gardens were a large source of Juglans regia seedlings, frequency of which decreased with the distance from villages (no species escaped from the historical park). Weed risk assessment revealed potential invasion danger only for Amorpha fruticosa.

First report of leaf blight caused by Phytophthora ramorum on cherry laurel (Prunus laurocerasus) in Washington State, USA.

In April 2014, Phytophthora ramorum (Werres, De Cock & Man in't Veld) was recovered from symptomatic foliage of cherry laurel (Prunus laurocerasus) at an ornamental plant nursery in Washington State. Cherry laurel, also known as English laurel, is widely propagated in WA because it is commonly used in landscaping. It is invasive in forests near the urban/wildland interface in the western US and in Europe (Rusterholz et al. 2018). Given its popularity as an ornamental species, the potential of this host to spread P. ramorum is of regulatory concern due to possible long distance spread to other states via nursery stock. Foliar symptoms consisted of dark brown lesions near wounds or around leaf margins where water collected. Shot-hole symptoms characterized by abscission zones and dropping of infected tissues were also observed. Lesions expanded beyond the margin of the shot-hole in some cases (Figure S1A). Phytophthora was isolated from symptomatic foliage by surface-sterilizing leaf pieces in 0.6% sodium hypochlorite and 2 rinses in sterile water. They were plated on PARP medium (Ferguson and Jeffers 1999). After 2-3 days, a slow-growing dense colony with coralloid hyphae was isolated onto V8 agar. P. ramorum was identified by observing morphological features (Figure S1B). Colony and spore morphology matched that of P. ramorum (Werres et al. 2001). The isolate was confirmed as P. ramorum by PCR and sequencing of ITS and COX1 regions using primers ITS1/ITS4 (White et al. 1990) and COX1F1/COX1R1 (Van Poucke et al. 2012). Sequences were submitted to GenBank (accession nos. ITS MT031969, COX1 MT031968). BLAST results showed at least 99% similarity with sequences of P. ramorum (ITS, KJ755124 [100%]; COX1, EU124926 [99%]). Multilocus genotyping with microsatellite markers placed the isolate in the EU1 clonal lineage. Pathogenicity of P. ramorum on cherry laurel was confirmed by completing Koch's Postulates using the isolate taken from this host. Two trials were done in a biocontainment chamber (USDA-APHIS permit # 65857) since P. ramorum is a quarantine pathogen and greenhouse trials could not be conducted, using detached stems from mature, visibly healthy cherry laurel plants growing in a landscape. Phytophthora ramorum inoculum was grown on V8A plates at 20®C for 2 weeks until sporangia were abundant. A zoospore suspension was produced by flooding plates with 7 ml sterile water, incubating for 2 hours at 5®C, then 1 hour at 24®C. Zoospores were observed with light microscopy, quantified with a hemocytometer and diluted to 1 x 104 zoospores/ml. A 10 µl droplet was placed at 3 wounded and 3 unwounded sites on 4 leaves per branch. In addition, a set of samples was inoculated by dipping foliage into the zoospore suspension for 30 seconds. A set of controls was mock inoculated using sterile water. Four branches per inoculation treatment were used and the trial was repeated once. Inoculated plant materials were incubated in moist chambers for 3-5 days at 20®C. Free moisture was present on foliage upon removal. Symptom development was assessed after incubation in the biocontainment chamber at 20®C for 7 days (Figure S1C). Phytophthora ramorum was reisolated from symptomatic tissue and the recovered culture was verified morphologically and by PCR and sequencing. It was isolated more often from foliage dipped in zoospore suspension than droplet inoculated, and more from wounded than unwounded sites. None of the water-inoculated controls were positive for P. ramorum. The presence of P. ramorum was also confirmed with DNA extraction from surface-sterilized symptomatic foliage followed by PCR and sequencing of the COX1 gene (EU124926, 100%) (Figure S2). To our knowledge, this is the first report of P. ramorum naturally infecting cherry laurel in the United States. Acknowledgements This work was supported by the USDA National Institute of Food and Agriculture, McIntire-Stennis project 1019284 and USDA APHIS Cooperative Agreement AP17PPQS&T00C070 Literature cited Ferguson and Jeffers, 1999. Plant Disease 83:1129-1136 Van Poucke, K. et al. 2012. Fungal Biology 116: 1178-1191. http://dx.doi.org/10.1016/j.funbio.2012.09.003 Werres, S. et al. 2001. Mycol. Res. 105:1155-1165. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.