Pseudodidymella Fagi in Slovenia: First Report and Expansion of Host Range

Article Pseudodidymella fagi in Slovenia: First Report and Expansion of Host Range

Nikica Ogris * , Ana Brglez and Barbara Piškur Department of Forest Protection, Slovenian Forestry Institute, 1000 Ljubljana, Slovenia * Correspondence: [email protected]

Received: 25 July 2019; Accepted: 19 August 2019; Published: 21 August 2019

Abstract: The fungus Pseudodidymella fagi is spreading in Europe and causing leaf blotch of European beech, Fagus sylvatica. Between 2008 and 2017, outbreaks of P. fagi were observed on European beech in Switzerland, Germany (also on F. orientalis), Austria, and Slovakia. In Slovenia, leaf blotch symptoms were first observed on F. sylvatica in 2018. P. fagi was identified as the causal agent of the observed symptoms in Slovenia by morphological examinations together with sequencing of the internal transcribed spacer (ITS) region of the rDNA. This study links the fungus to the expansion of the known distribution of the disease to Slovenia, and based on in vitro pathogenicity trials, also to a new potential host, Quercus petraea. The pathogenicity tests confirmed F. sylvatica and F. orientalis as hosts for P. fagi, but not Castanea sativa, where pathogenicity to F. orientalis was proved for first time in vitro. Although Koch’s postulates could not be proven for C. sativa, it seems to be partially susceptible in vitro because some of the inoculation points developed lesions. Additionally, damage to Carpinus betulus related to P. fagi near heavily infected beech trees was observed in vivo but was not tested in laboratory trials. Based on the results and our observations in the field, it is likely that P. fagi has a wider host range than previously thought and that we might be witnessing host switching.

Keywords: Pycnopleiospora fagi; leaf blotch; pathogenicity test; inoculation test; Fagus orientalis; Fagus sylvatica; Quercus petraea; mycopappus-like propagule; Carpinus betulus; host switching

1. Introduction Pseudodidymella fagi C.Z. Wei, Y. Harada & Katum. is an ascomycete that was described in Japan in 1993, where it was associated with leaf blotch of Japanese beech, Fagus crenata Blume [1]. The sexual morph is formed over winter, and ascospores mature in late spring and initiate new infections of young beech leaves. Its pycnopleiospora asexual morph is formed under favourable conditions on necrotic leaf spots a few weeks after the initial infection has occurred. Conidia are responsible for secondary infections in summer [1]. The first observation of the pathogen in Europe was in Switzerland in 2008, where it was identified on European beech (Fagus sylvatica L.) as a new host [2]. In 2016, P. fagi was also identified in southern Germany on F. sylvatica and on Oriental beech (Fagus orientalis Lipsky) in the Munich Botanical Garden [2] and in Austria [3]. In 2017, the disease was recorded in Slovakia [4]. Reports of P. fagi spread in Europe indicate that it possesses the fundamental characteristics of an invasive alien species. However, this hypothesis still needs further testing [2]. In 2018, leaf blotch of European beech was observed in central Slovenia. Leaves of F. sylvatica showed dark brown, irregularly shaped or confluent necrotic spots. Necrotic spots were more numerous on young plants and the lower branches of mature trees. The intensity of leaf blotch decreased progressively with increasing distance from the ground. To identify the causal agent, morphological and molecular analyses together with pathogenicity trials on F. sylvatica were used to identify and confirm the pathogen. In addition, pathogenicity tests were performed on other

Forests 2019, 10, 718; doi:10.3390/f10090718 www.mdpi.com/journal/forests Forests 2019, 10, 718 2 of 10 representative species of the Fagaceae family, i.e., F. orientalis, Castanea sativa Mill., and Quercus petraea (Matt.) Liebl., to test the possibility of a wider host range of P. fagi.

2. Materials and Methods

2.1. Sample Collection and Distribution of Pseudodidymella fagi Leaves of F. sylvatica infected by P. fagi were found and collected in August 2018 in Kamniška Bela, in central Slovenia (46.32020◦ N, 14.60161◦ E, 620 m a.s.l.). The infected leaves were collected and deposited in the Mycotheca and Herbarium of the Slovenian Forestry Institute (LJF 7014–LJF 7016). In addition, further collection of European beech leaves from the leaf litter was done in spring 2019 to identify the sexual morph (LJF 7017). Visually healthy leaves from representatives of the Fagaceae family were collected on 23 August 2018, for pathogenicity trials (Table1).

Table 1. Locations of samples collected for pathogenicity trials.

Plant Species Location Latitude (◦ N) Longitude (◦ E) Elevation (m a.s.l.) Fagus sylvatica Ljubljana 46.05195 14.47859 312 Fagus orientalis VolˇcjiPotok 46.18361 14.61061 384 Quercus petraea Ljubljana 46.05277 46.05277 322 Castanea sativa Ljubljana 46.05257 14.47903 319

The Slovenia Forest Service (SFS) was used to report symptoms and send samples to conﬁrm the pathogen and to determine its distribution in Slovenia.

2.2. Morphological Analyses Symptomatic leaves were examined, photo-documented, and analyzed with an Olympus SZX16 stereomicroscope (Tokyo, Japan) with an Olympus UC90 camera (Münster, Germany), an Olympus BX53 microscope (Tokyo, Japan) with an Olympus DP26 camera (Tokyo, Japan), and an Olympus CellSens Standard v1.18 software. Isolations from single or multiple conidium and/or ascospore were carried out by aseptically transferring single propagules from leaves to fresh malt-extract agar plates (MEA; 2% malt extract, 1.5% agar; Becton Dickinson, Sparks, MD, USA) and to potato dextrose agar plates (PDA; 3.9% potato dextrose agar; DifcoTM, Becton Dickinson, Sparks, MD, USA) with the tip of a scalpel and incubating them at 24 ◦C in the dark. In total, ten isolates from fruiting bodies were acquired. Representative isolates were deposited in the culture collection of the Laboratory of Forest Protection (Slovenian Forestry Institute, Ljubljana, Slovenia, ZLVG683 and ZLVG684). For isolations from lesions on leaves, a procedure similar to that used by Gross, Beenken, Dubach, Queloz, Tanaka, Hashimoto and Holdenrieder [2] was used—leaves were surface sterilized for 30 s in 80% ethanol, and squares of approximately 5 mm2 of symptomatic tissue per leaf were excised and 1 transferred to MEA plates amended with 100 mg L− streptomycin (Sigma-Aldrich, St. Louis, MO, USA).

2.3. DNA Extraction, DNA Sequencing, and Identification Genetic analyses were used to confirm the identity of the fungal propagules and obtained isolates. DNA was extracted from the mycelium, scraped from the MEA agar plates, or was extracted from 3–6 conidiomata carefully detached directly from infected leaves. Genomic DNA was extracted with a commercial NucleoSpin® Plant II kit (Macherey Nagel, Düren, Germany) following the manufacturer’s protocol, after homogenizing the fungal material with a lysing matrix A tube (MP Biomedicals, Solon, OH, USA) using a Precellys Evolution device (Bertin Technologies, Montigny-le-Bretonneux, France). The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was amplified with primer Forests 2019, 10, 718 3 of 10 pair ITS1 and ITS4 [5]. The final volume of the amplification mixtures was 50 µL and contained 25 µL AmpliTaq Gold® 360 Master Mix (Applied Biosystems, Foster City, CA, USA), 1 µL 360 GC Enhancer (Applied Biosystems), 3 µL each of 10 µM primers (Sigma-Aldrich), 4 µL of approximately 10 µg/mL extracted DNA, and 14 µL of nuclease-free water. The PCR conditions were as follows: 95 ◦C for an initial 10-min denaturation, 35 cycles of 94 ◦C for 1 min, annealing at 61.5 ◦C for 1 min and extension at 72 ◦C for 1 min, and a final extension at 72 ◦C for 10 min. The obtained PCR products were cleaned using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and sequenced at a sequencing facility (Eurofins, Ebersberg, Germany) in both forward and reverse directions using the same primers as for the PCRs. Sequences were visualised and manually edited using Geneious Prime® v.2019.0.3. (Biomatters Ltd., Auckland, New Zealand). Each sequence was used to perform individual nucleotide–nucleotide searches with the megablast algorithm from the NCBI website on 12 July 2019 [6]. Representative sequences were deposited in the GenBank database.

2.4. Pathogenicity Tests Pathogenicity tests were performed on representative tree species from the family Fagaceae: Fagus sylvatica, Fagus orientalis, Castanea sativa, and Quercus petraea. Visually healthy leaves of F. sylvatica, C. sativa, and Q. petraea were collected in mixed broadleaved forests on 23 August 2018 (Table1). The leaves were rinsed with autoclaved distilled water and placed on moistened paper tissues in sterile glass plates (19 cm diameter, 4 cm height) as follows: four beech leaves per plate, four oak leaves per plate, and one sweet chestnut leaf per plate for a total of eight leaves per host. For inoculation, firstly three to six P. fagi propagules on symptomatic F. sylvatica leaves were morphologically and molecularly identified, and secondly the remaining propagules from infected F. sylvatica leaves were carefully transferred with a sterile scalpel to the upper side of the leaves, which were included in the pathogenicity trials. Leaves of F. sylvatica, F. orientalis, and Q. petraea were inoculated with five propagules and C. sativa leaves with ten propagules. In total, there were 40 inoculation points for F. sylvatica, F. orientalis, and Q. petraea, and 80 inoculation points for C. sativa. Four non-inoculated healthy leaves of each species represented controls. After inoculation, leaves were incubated in petri dishes under daylight conditions at room temperature (22–25 ◦C). Paper tissues were periodically remoistened with sterile distilled water to avoid desiccation. The leaves were regularly inspected using an Olympus SZX16 stereomicroscope to monitor the infection process. After 22 days of incubation, re-isolations were carried out from the freshly developing conidiomata and from necrotic leaf tissues developing in the immediate vicinity of the inoculation points. Additional isolations from non-symptomatic leaves and controls were carried out to verify that no latent P. fagi infections were present. For isolations from freshly developing conidiomata and for re-isolations from leaves, the same procedures were used as described in morphological analyses in Section 2.2. The following number of samples were used for re-isolations per host: 36 samples for F. sylvatica, 31 samples for F. orientalis, 21 samples for C. sativa, and 18 samples for Q. petraea. Plates were incubated at 24 ◦C in the dark and regularly checked for mycelium growth for two weeks (negative controls for 29 days). Any outgrown mycelium was immediately transferred to fresh PDA and MEA plates. The isolates were grouped into morphotypes based on colony characteristics, and representative isolates from each morphotype were identified based on the ITS rDNA sequence.

3. Results

3.1. Distribution and Identification of Pseudodidymella fagi The first symptoms of P. fagi on European beech in Slovenia were observed in August 2018 in Kamniška Bela (46.32020◦ N, 14.60161◦ E) and in Ljubljana (46.05195◦ N, 14.47859◦ E). Both sites were located near streams in mixed managed forests and were characterized by high humidity. With the help of foresters from the Slovenia Forest Service, the disease was confirmed to be present in a large part of Slovenia (Figure1). Forests 2019, 10, 718 4 of 10 Forests 2019,, 10,, xx FORFOR PEERPEER REVIEWREVIEW 4 of 10

Figure 1. Distribution map of Pseudodidymella fagi inin Slovenia Slovenia by by regional regional units units of of the the Slovenia Slovenia ForestForest Service along withwith locationslocations thatthat werewere checked for presencepresence ofof thethe pathogen.pathogen.

Leaves ofofF. F. sylvatica sylvaticashowed showedshowed dark darkdark brown brownbrown necrotic necroticnecrotic spots(Figure spotsspots 2(Figure(Figurea). Necrotic 2a).2a). spotsNecroticNecrotic were spotsspots irregularly werewere shapedirregularlyirregularly or conﬂuent shapedshaped oror and confluentconfluent usually hadandand ausuallyusually sharp and hadhad darker aa sharpsharp border andand withdarkerdarker healthy borderborder tissues withwith (Figurehealthyhealthy2 b)tissuestissues and with(Figure(Figure mycopappus-like 2b)2b) andand withwith mycopappus-likemycopappus-like propagules (asexual propagulespropagules morph) (asexual(asexual on the surface morph)morph) (Figure onon thethe2c). surfacesurface The propagules (Figure(Figure 2c).2c). were TheThe globose,propagules multicellular, were globose, hyaline, multicellular, or very hyaline, pale yellow, or very measuring pale yellow, 142–239 measuring (ø = 193)142–239µm (ø in = diameter 193) μm (withoutinin diameterdiameter appendages; (without(without appendages;appendages; Figure2d). They FigureFigure bore 2d).2d). numerous TheyThey borebore ( > numerousnumerous130) hyaline, (>130)(>130) unbranched, hyaline,hyaline, unbranched,unbranched, up to 4-septate, upup 84–165toto 4-septate,4-septate, (ø = 129)- 84–16584–165µm long,(ø(ø == 129)-129)- andμ 4.3–6.4m long, (ø =and5.2)- 4.3–6.4µm wide (ø = appendages 5.2)-μm wide (Figure appendages2e) originating (Figure from 2e) globular,originating monilioid, from globular, hyaline, monilioid, or pale yellow hyaline, hyphae, or pale which yellow measured hyphae, 8.6–16.1 which (ø measured= 11.6) µm 8.6–16.1 in diameter (ø = (Figure11.6) μm2f,g). in diameter (Figure 2f,g).

Figure 2. SymptomsSymptoms and morphology of Pseudodidymella fagi:(:: ((a,,b)) NecroticNecrotic leaf leaf spots; spots; ( (c)) necrotic necrotic leaf leaf spotspot coveredcovered with with mycopappus-like mycopappus-like propagules; propagules; (d) macroscopic (d)) macroscopicmacroscopic view of mycopappus-likeviewview ofof mycopappus-likemycopappus-like propagule, conidium;propagule, ( econidium;) septate appendages; (e)) septateseptate appendages;appendages; (f) basal cells ((ff) of) basalbasal an appendage; cellscells ofof anan appendage;appendage; (g) globular (( cellsg)) globularglobular in the central cellscells inin part thethe ofcentral a propagule; part of a ( hpropagule;) ascomata ( onh)) upperascomataascomata leaf onon surface; upperupper ( ileafleaf) ostiole surface;surface; of an ((ii)) ascoma; ostioleostiole of (ofj) anan asci; ascoma;ascoma; (k) ascospores. ((jj)) asci;asci; ((k)) ascospores.

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On 19 April 2019, at the time of European beech leaf flush, the sexual morph of P. fagi was observedOn 19 in April the 2019,litter aton the F. timesylvatica of European leaves from beech the leaf previous flush, the year. sexual Ascomata morph of wereP. fagi darkwas brown observed to inblack the litter(Figure on F.sylvatica2h,i), subgloboseleaves from to lenticular, the previous semi year.-immersed Ascomata in the were host dark tissue, brown measuring to black (Figure 121–2402h,i), (ø subglobose= 178) μm in to diameter lenticular, with semi-immersed a sunken centre in the in host dry tissue, state. measuring Asci were121–240 51–83 (ø (ø = =63)178) μmµ mlongin diameterand 9.1– with11.6 (ø a sunken= 10.2) μ centrem wide, in dry bitunicate, state. Asci clavate were to 51–83 cylindrical, (ø = 63) withµm longround and apex, 9.1–11.6 short ( østalked= 10.2 )(Figureµm wide, 2j). bitunicate,Ascospores clavatewere 17.4–25.5 to cylindrical, (ø = 21.6) with μm long round and apex, 4.0–6.5 short (ø = stalked5.5) μm wide, (Figure hyaline,2j). Ascospores mostly 1-septate, were 17.4–25.5constricted (ø =at 21.6)the septum,µm long distinctly and 4.0–6.5 pointed (ø = 5.5) butµ mrounded wide, hyaline,at the apex mostly (Figure 1-septate, 2k). Morphological constricted at theidentification septum, distinctly corresponded pointed to the but description rounded at of the Pseudodidymella apex (Figure 2fagik). [1,2,4,7]. Morphological identification correspondedThe P. fagi to cultures the description on PDA of werePseudodidymella pulvinate, olivaceo fagi [1,2us-grey,4,7]. with black exudates on the upper side Theand P.dark fagi olivaceouscultures on to PDA blackish were on pulvinate, the reverse. olivaceous-grey The P. fagi colonies with black on MEA exudates were on mostly the upper flat, sideblackish and darkto dark olivaceous olivaceous, to blackish patchily on fl theoccose reverse. and Thegreyish.P. fagi Thecolonies growth on rate MEA of were P. fagi mostly colonies flat, blackishmeasured to 33–54 dark olivaceous, (ø = 42) mm patchily in 21 days floccose at 24 and °C in greyish. the dark The on growth PDA. rate of P. fagi colonies measured 33–54The (ø = morphological42) mm in 21 daysidentification at 24 ◦C inof theboth dark fungal on PDA.propagules and isolated cultures was confirmed basedThe on morphological ITS rDNA sequence identification comparison. of both The fungal sequences propagules from and propagules isolated cultures(MN170774) was confirmed and from basedmycelial on isolates ITS rDNA ZLVG683 sequence (MN170775) comparison. and ZLVG684 The sequences (MN170776) from propagules showed 100% (MN170774) identity and(448 frombp/448 mycelial bp) with isolates the sequence ZLVG683 obtained (MN170775) from P. fagi and holotype ZLVG684 H2579 (MN170776) (GenBank showed Acc. No. 100% LC150787). identity (448 bp/448 bp) with the sequence obtained from P. fagi holotype H2579 (GenBank Acc. No. LC150787). 3.2. Pathogenicity Tests 3.2. Pathogenicity Tests 3.2.1. Fagus sylvatica 3.2.1. Fagus sylvatica No necrotic lesions developed on negative controls of F. sylvatica (Figure 3a). On inoculated leavesNo of necrotic F. sylvatica lesions, necrotic developed lesions on started negative to develop controls after of F. sylvaticatwo days(Figure underneath3a). On or inoculated in the immediate leaves ofvicinityF. sylvatica of the, necrotic inoculated lesions propagule started to (Figure develop 3c after), and two microscopic days underneath examination or in the showed immediate numerous vicinity ofappressoria the inoculated on the propagule leaf surface (Figure (Figure3c), and3e). microscopicAfter 21 days, examination 70% of inoculations showed numerous had a typical, appressoria light to ondark the brown leaf surface necrosis (Figure under3e). the After propagule 21 days, (Figure 70% of 3b inoculations). Fourteen haddays a typical,after inoculation, light to dark numerous brown necrosismycopappus-like under the propagulepropagules (Figure were observed3b). Fourteen on 10 days out after of the inoculation, 30 lesions numerousobserved mycopappus-like(Figure 3d). From propagules12 isolations were of mycopappus-like observed on 10 out propagules, of the 30 lesionsfive isolates observed similar (Figure to 3P.d). fagi From were 12 obtained. isolations Re- of mycopappus-likeisolations from 21 propagules, lesions of F. five sylvatica isolates yielded similar seven to P. fagiisolateswere (out obtained. of 34) morphologically Re-isolations from resembling 21 lesions ofP. F.fagi sylvatica (Figureyielded 3g–i) and seven 27 isolatesother isolat (outes of grouped 34) morphologically into seven morphoty resemblingpes.P. The fagi (FigureITS sequence3g–i) and of the 27 otherrepresentative isolates grouped isolate of into the seven P. fagi morphotypes. morphotype (MN170779) The ITS sequence obtained of the from representative symptomatic isolate F. sylvatica of the P.leaves fagi morphotype was 100% identical (MN170779) with obtainedP. fagi sequence from symptomatic LC150787. TheF. sylvatica most frequentlyleaves was isolated 100% identical fungus from with P.necrosis fagi sequence on inoculated LC150787. F. Thesylvatica most frequentlyleaves was isolated Colletotrichum fungus from sp., necrosis which onwas inoculated obtainedF. from sylvatica 13 leavesisolations was (outColletotrichum of 34). sp., which was obtained from 13 isolations (out of 34).

Figure 3. Cont.

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FigureFigure 3.3. PathogenicityPathogenicity testtest onon FagusFagus sylvaticasylvatica:(:: ((aa)) ControlControl onon thethethe 21st21st21st day;day;day; (((bb))) necroticnecroticnecrotic leafleafleaf spotsspotsspots ononon thethethe 21st21st21st day;day;day; (((ccc))) aaa lesionlesionlesion developeddevelopeddeveloped underunderunder aaa propagulepropagulepropagule ononon thethethe 2nd2nd2nd day;day;day; ((d(d))) fresh freshfresh mycopappus-like mycopappus-likemycopappus-like propagulespropagules developeddeveloped on a a necrotic necrotic spot spot on on the the 14th 14th day; day; (e) () e appressoriumappressorium) appressorium formationformation formation (arrows)(arrows) (arrows) onon the onthe the11th 11th day; day; (ff)) disintegrationdisintegration (f) disintegration ofof thethe of theinoculatedinoculated inoculated propagulepropagule propagule onon thethe on 21st21st the 21st day;day; day; ((g,,h) () g upperupper,h) upper andand andreversereverse reverse sideside sideof culture of culture re-isolated re-isolated from fromnewly newly formed formed mycop mycopappus-likeappus-like propagule propagule on PDA, on PDA,respectively; respectively; (ii)) re-re- (isolationisolationi) re-isolation fromfrom newly fromnewly newly formedformed formed mycmycopappus-like mycopappus-like propagule propagule on MEA. on MEA.

3.2.2.3.2.2. FagusFagus orientalisorientalis NoNo necroticnecrotic lesions lesions developed developed on on negative negative controls controls of F. of orientalis F. orientalis(Figure (Figure(Figure4a). On 4a).4a). inoculated OnOn inoculatedinoculated leaves ofleavesleavesF. orientalis ofof F. ,orientalis lesions, started, lesionslesions to startedstarted develop toto after developdevelop two daysafterafter (Figure twotwo daysdays4c). Altogether,(Figure(Figure 4c).4c).74% Altogether,Altogether, of inoculations 74%74% ofof developedinoculationsinoculations necrosis developeddeveloped directly necrosisnecrosis underneath directlydirectly or underneathunderneath near the inoculated oror nearnear thethe propagules. inoculatedinoculated propagules.propagules. The size of theTheThelesions sizesize ofof graduallythethe lesionslesions increased graduallygradually (Figure increasedincreased4b,d). (Figure(Figure Newly 4b,d).4b,d). formed NewlyNewly mycopappus-like formedformed mycopappus-likemycopappus-like propagules propagulespropagules started to startedstarted develop toto fromdevelop three from lesions three after lesions 14 days after (Figure 14 days4e). (Figure Five pure 4e). Five cultures pure were cultures obtained were from obtained isolations from ofisolations freshly formedof freshly mycopappus-like formed mycopappus- propagules,likelike propagules,propagules, similar to P.similarsimilar fagi. Re-isolationstoto P. fagi.. Re-isolationsRe-isolations from symptomatic fromfrom symptomaticsymptomatic tissues of F.tissuestissues orientalis ofof F.yielded orientalis two yieldedyielded pure twotwo cultures purepure culturescultures (out of 30) (out(out morphologically ofof 30)30) morphologicallymorphologically resembling resemblingresemblingP. fagi P.(Figure fagi (Figure(Figure4f,g). The4f,g). ITS The sequence ITS sequence of the of representative the representative isolate isolate of the ofP. the fagi P.morphotype fagi morphotypemorphotype (MN170777) (MN170777)(MN170777) obtained obtainedobtained from symptomaticfromfrom symptomaticsymptomaticF. orientalis F. orientalisleaves was leavesleaves 100% waswas identical 100%100% identicalidentical to P. fagi sequencetoto P. fagi LC150787.sequencesequence LC150787.LC150787. The most frequently TheThe mostmost isolatedfrequentlyfrequently fungus isolatedisolated from fungusfungusF. orientalis fromfrom leavesF. orientalis was Colletotrichum leavesleaves waswas Colletotrichumsp. (18 outof sp.sp. 30 (18(18 isolates). outout ofof 3030 isolates).isolates).

FigureFigure 4.4. PathogenicityPathogenicity test test on onFagus Fagus orientalis orientalis:(::a (()a Control)) ControlControl on onon the thethe 21st 21st21st day; day;day; (b) (( necroticb)) necroticnecrotic leaf leafleaf spots spotsspots on the onon 21stthethe 21st21st day; day;day; (c) a ((c lesionc)) aa lesionlesion developed developeddeveloped under underunder a propagule aa propagulepropagule on the onon 2ndthethe 2nd2nd day; day;day; (d) lesion((d)) lesionlesion under underunder a propagule aa propagulepropagule on theon the 11th 11th day; day; (e) newly(e)) newlynewly formed formedformed mycopappus-like mycopappus-likemycopappus-like propagule propagulepropagule on a on necrotic a necrotic spot spot on the on 14th the 14th day; (day;f,g) re-isolation((ff,,g)) re-isolationre-isolation from from newlyfrom newlynewly formed formedformed mycopappus-like mycopappus-mycopappus- propagulelikelike propagulepropagule on MEA onon MEAMEA and PDA, andand PDA,PDA, respectively. respectively.respectively.

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3.2.3.3.2.3. QuercusQuercusQuercus petraeapetraea NoNo necroticnecrotic lesionslesions developeddeveloped onon negativenegative controlscontrols ofof Q. Q. petraeapetraea (Figure (Figure(Figure5 5a).a).5a). Lesions LesionsLesions developed developeddeveloped onon 28% 28% of of inoculation inoculationinoculation points, points, i.e., i.e.,i.e., directly directly underneath underneath or oror near near the the inoculated inoculated propagules propagules (Figure (Figure 55b,c). 5b,c).b,c). NoNo mycopappus-like mycopappus-like propagulespropagules developeddeveloped onon thethe lesionslelesionssions duringduring thethe experiment. experiment. Re-isolations Re-isolations from from symptomaticsymptomatic tissues tissuestissues of of Q.Q. petraeapetraea petraea yieldedyieldedyielded oneone one purepure pure cultureculture culture (out (out (out ofof of 10)10) 10) morphologicallymorphologically morphologically resemblingresembling resembling P.P. P.fagifagi fagi (Figure(Figure(Figure 5d,e)5d,e)5d,e) andand and ninenine nine isolates,isolates, isolates, whichwhich which werewere were groupedgrouped into into seven sevenseven morphotypes.morphotypes. TheThe ITSITS sequence sequencesequence ofof the the isolate isolate of of the the P. P.P. fagi fagi morphotype morphotype (MN170778) (MN170778) obtained obtained fromfrom symptomatic symptomatic Q. Q. petraea petraea leaves leaves was was 100%100% identicalidentical toto P.P. fagi fagifagi sequence sequencesequence LC150787. LC150787.LC150787. The The most most frequently frequently isolated isolated fungus fungus from from Q.Q. petraeapetraea inoculatedinoculated leavesleaves waswas Paraphaeosphaeria Paraphaeosphaeria sp.sp. (two(two outout ofof tenten isolates).isolates).

FigureFigure 5.5. PathogenicityPathogenicity test testtest on ononQuercus QuercusQuercus petraea petraeapetraea:(: :a ()(aa) Control) ControlControl on onon the thethe 21st 21st21st day; day;day; (b ) ((b necroticb)) necroticnecrotic leaf leafleaf spot spotspot on the onon the21stthe 21st21st day; day;day; (c) disintegration ((cc)) disintegrationdisintegration of the ofof inoculated thethe inoculatedinoculated propagule propagulepropagule on the onon 21st thethe day; 21st21st ( d day;day;,e) upper ((dd,e,e)) upperandupper reverse andand reversereverse side of sidecultureside of of culture re-isolatedculture re-isolated re-isolated from necrotic from from necrot necrot leaf spoticic leaf leaf on spot MEA,spot on on respectively. MEA, MEA, respectively. respectively.

3.2.4.3.2.4. CastaneaCastanea sativasativa NoNo necroticnecroticnecrotic lesions lesionslesions developed developeddeveloped on negativeonon negativenegative controls controlscontrols of C. sativa ofof C.C.(Figure sativasativa6 a).(Figure(Figure Only 19%6a).6a). ofOnlyOnly inoculation 19%19% ofof pointsinoculationinoculation developed pointspointslesions developeddeveloped (Figure lesionslesions6d) and (Figure(Figure most 6d)6d) of the andand inoculated mostmost ofof thethe leaves inoculatedinoculated stayed leaves healthyleaves stayedstayed (Figure healthyhealthy6b,c). (FigureNo(Figure mycopappus-like 6b,c).6b,c). NoNo mycopappus-likemycopappus-like propagules developed propagulespropagules on the developeddeveloped observed lesionsonon thethe on obseobse artiﬁciallyrvedrved lesionslesions inoculated onon artificiallyartificiallyC. sativa inoculatedleaves.inoculated Re-isolations C.C. sativasativa leaves.leaves. from developed Re-isolationsRe-isolations lesions fromfrom on developeddevelopedC. sativa lesionsleaveslesions yieldedonon C.C. sativasativa no cultures leavesleaves thatyieldedyielded would nono culturesmorphologicallycultures thatthat wouldwould resemble morphologicallymorphologicallyP. fagi, however resembleresemble yielded P.P. fagifagi nine,, howeverhowever isolates, yieldedyielded which nine werenine isolates,isolates, classiﬁed whichwhich into threewerewere classifiedmorphotypes.classified intointo Thethreethree most morphotypes.morphotypes. frequently isolated TheThe mostmost fungus frequentlyfrequently from C. isolatedisolated sativa leaves fungusfungus was fromfromGnomoniopsis C.C. sativasativa smithogilvyileavesleaves waswas GnomoniopsisL.A.Gnomoniopsis Shuttlew., smithogilvyismithogilvyi E.C.Y. Liew L.A.L.A. & D.I. Shuttlew.,Shuttlew., Guest. E.C.Y.E.C.Y. LiewLiew && D.I.D.I. Guest.Guest.

FigureFigure 6.6. PathogenicityPathogenicity testtest onon CastaneaCastanea sativasativa::( : ((aaa))) Control ControlControl on onon the thethe 21st 21st day;day; ((bb)) inoculatedinoculated leafleaf onon thethe 21st21st day; day; ((cc)) germinationgermination ofof aa propagulepropagule onon on thethe the 2nd2nd 2nd day;day; day; (( d(d))) necroticnecrotic necrotic leafleaf leaf spotspot spot onon on thethe the 21st21st 21st day.day. day.

Forests 2019, 10, 718 8 of 10

4. Discussion The disease symptoms caused by Pseudodidymella fagi on European beech were observed across a large part of Slovenia, which is now the fifth European country with a known presence of P. fagi. Susceptible hosts for P. fagi are known to be F. crenata, F. sylvatica, and F. orientalis, but the pathogenicity tests conducted in this study proved that P. fagi is also a potential pathogen for Q. petraea. Therefore, this study is the first confirmation of Koch’s postulates for P. fagi towards F. orientalis and Q. petraea. It is still not clear whether P. fagi is an overlooked pathogen that is causing more evident damage throughout Europe due to climatic changes or a newly introduced invasive species [2]. It is probable that the similar symptomatology compared to Apiognomonia errabunda (Roberge ex Desm.) Höhn. concealed the earlier discovery of P. fagi in Slovenia. Numerous recent reports of P. fagi occurring in Europe support the hypothesis that P. fagi was introduced into Europe, most likely from Asia [1,7], but further genetic studies are needed to appropriately address this question. Visually healthy leaves of the four tree species were inoculated with propagules of P. fagi. Even though 19–74% of the inoculation spots developed lesions under or in the immediate vicinity of the propagule, re-isolations from those regions did not always yield the culture of P. fagi. Furthermore, re-isolation of P. fagi after artificial infection proved to be difficult and resulted in a very low success rate for all tested species. On the other hand, numerous other fungi were re-isolated from the necrotic leaf spots under the inoculation points, such as Colletotrichum sp. from European beech and Oriental beech, Paraphaeosphaeria sp. from sessile oak, and Gnomoniopsis smithogilvyi from sweet chestnut. Some of the identified species are well-known endophytes [8–11]. We speculate that endophytes could have an important role in competing with P. fagi for the same substrate or could have a role in the development of necrosis. Additionally, endophytes could play a decisive role in combating the future spread of P. fagi across Europe, as is also known for other endophytic fungi [12,13]. As such, the role of endophytes in the development and spread of the disease caused by P. fagi should be investigated in the future. Our study reports the first finding of P. fagi in Slovenia and its further spread in Europe towards the south. We confirmed the pathogenicity of P. fagi towards F. sylvatica. This is the first study that confirmed F. orientalis as a host, in agreement with Koch’s postulates. Our in vitro inoculation experiment showed potential pathogenicity towards Q. patraea and C. sativa, which is the first report of new potential hosts for P. fagi in Europe. However, no characteristic symptoms for P. fagi were observed in the field on Q. petraea and C. sativa leaves. Re-isolations of the pathogen from inoculated C. sativa leaves were not successful. However, C. sativa seems to be partially susceptible in vitro because 19% of inoculation points developed lesions. Koch’s postulates could not be fulfilled for C. sativa; nevertheless, it is possible that the observed necroses were caused by P. fagi and its re-isolation failed because re-isolations of P. fagi proved to be difficult for all tested species. In the field, numerous heavily damaged leaves of common hornbeam (Carpinus betulus L.) were observed in the immediate vicinity of European beech heavily infected by P. fagi. Macroscopic examination showed that some necrotic lesions on C. betulus leaves developed underneath or in the immediate vicinity of the mycopappus-like propagules of P. fagi. However, no newly developed propagules were found on those lesions. We hypothesize that C. betulus could be another possible host, where P. fagi acts as a weak pathogen. Therefore, extended pathogenicity tests should be performed that would include representatives from the Fagales order, i.e., from the following genera: Alnus, Betula, Carpinus, Corylus, and Ostrya. We are most likely witnessing host jumps and host switching of P. fagi [2,7,14], and close monitoring of this arising pathogen throughout Europe would clarify the situation of the disease on other hosts.

5. Conclusions P. fagi is reported for first time in Slovenia. The pathogenicity of P. fagi towards F. sylvatica was confirmed once again. Pathogenicity towards F. orientalis and Q. petraea was confirmed for first time in vitro. C. sativa seems to be partially susceptible in vitro. Based on the results and our observations Forests 2019, 10, 718 9 of 10 in the field, it is likely that P. fagi has a wider host range than previously thought and that we are most likely witnessing host jumps and host switching of P. fagi.

Author Contributions: Conceptualization, N.O.; data curation, N.O., A.B., and B.P.; formal analysis, N.O. and B.P.; funding acquisition, N.O.; investigation, N.O., A.B., and B.P.; methodology, N.O. and B.P.; project administration, N.O.; resources, N.O., A.B., and B.P.; supervision, N.O.; Validation, N.O., A.B., and B.P.; visualization, N.O.; writing—original draft, N.O.; writing—review and editing, N.O., A.B., and B.P. Funding: This research was funded by the Ministry of Agriculture, Forestry, and Food (Public Forestry Service) and the Slovenian Research Agency (Research Program P4-0107). Acknowledgments: We are grateful to Špela Jagodic and Zina Devetak for technical assistance and to foresters from the Slovenia Forest Service who helped with providing samples and information about the distribution range of the disease in Slovenia. We thank Thomas Cech at the Austrian Research Centre for Forest in Vienna for providing initial help and encouragement for searching for the disease. We are grateful to Jan Nagel for providing language help. We are especially grateful to two reviewers for their constructive comments, which improved the manuscript greatly. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

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