# Characterization of a Novel Emaravirus Affecting Ash Species (_Fraximus_ spp.) in Europe
## 2 Materials and Methods  
### Plant Material and RNA Extraction  
Following observations of suspected virus symptoms in _F. excelsior_ in Switzerland, other symptomatic trees, including manna ash (_Fraxinus ornu_ L.), were found at different geographical locations across Europe. These included trees in both managed semi-natural ecosystems and urban environments, independent of latitude and elevation. Affected ash exhibited chlorotic ringspots, mottle and leaf deformations, ranging from curling to a severe narrowing of the leaflets termed shoestring. Cauliflower galls--growths caused by the gall mite _Aceria fraxinivora_ were also recorded on some of the symptomatic trees. A selection of symptoms documented in _F. excelsior_ from Basel, Switzerland in 2016 and _F. ornu_ from Hamburg, Germany in 2018 are shown in Figure 1.  
For HTS analyses, total RNA was extracted as described by [PERSON] 2007 [26], excluding the phenol/chloroform step, from leaf material collected in 2016 from two _Fraxinus excelsior_ trees (E55262 and E55268, Basel, Switzerland), displaying virus-like disease symptoms thought to be caused by virus infection. Leaf samples displayed chlorotic ringspots, lines and leaf deformations including a severe narrowing of the leaflets, termed shoestring. RNA was also extracted from a second set of pooled leaf samples (E57321-E57325, E57328-E57330, E57335-E57337, E57339) originating from 12 _Fraxinus ornu_ trees (Hamburg, Germany, sampled in 2018) displaying identical symptoms as the ash trees in Basel. The same total RNA extraction method was also applied to the following samples collected from locations across Europe between 2016 and 2019: E55270 (_F. excelsior_, Basel, Switzerland, sampled 2016), E56800, E56802-E56814, E56818, E56820 (_F. excelsior_, Dalby Soderskog, Sigtuna, Bromma, Vreta, Sweden, sampled 2017), E57294, E5748, E57480, E57481 (_F. excelsior_, Hamburg, Germany, sampled 2018), E57298, E57299, E57331, E57332, E57326, E57338, E57482, E57483, E57485-E57495, E57515-E57519, (_F. ornu_  
Figure 1: Leaf symptoms observed in virus-infected _Fraxinus_ spp. and associated gall mites. **(A)** Shoestring (top) in comparison to normal shaped _F. excelsior_ leaf (bottom), (E552628) **(B)** Leaf of _F. excelsior_ (E55270) exhibiting deformation chlorotic lines and mottle, **(C)**_F. ornu_ (E57322) leaves with vein yellowing and shoestring, **(D)** deformed leaflets of _F. ornu_ (E57335) exhibiting chlorotic ringspots and mottle. Cauliflower galls (yellow arrows) observed in the _F. ornu_ canopy **(E)**. Electron microscopic image of gall mite _A. fraxinivora_ prepared from a cauliflower gall **(F)**.  
Hamburg, Germany, sampled 2018), E57471, E57472 (_F. ornus_ Bolzano, Italy, sampled 2018), E57455, E57456-E57459, E57465-E57469 (_F. excelsior_, Sweden, sampled 2018), E57340 (_F. excelsior_ Crans Montana, Switzerland, sampled 2018), E58222, E58223, E58235-E58246 (_F. excelsior_ Krusenstern Herrgard, Hallekis Blomberg, Vasterplana, Sweden sampled 2019), E58250 (_F. excelsior_ Berlin, Germany, sampled 2019), E58592-E58598 and E58047 (_F. ornus_ Hamburg, Germany, sampled 2019). Samples were collected from trees in urban locations, with both symptomatic and asymptomatic leaves being collected for analysis. Eleven _F. ornus_ samples (E57504-E57514) were also collected in 2018 in Hamburg from symptomatic and asymptomatic trees at a local tree nursery and the same methodology was applied.