Effects of Verticillium nonalfalfae on Ailanthus altissima and associated indigenous and invasive tree species in eastern Austria

Effects of Verticillium nonalfalfae on Ailanthus altissima and associated indigenous and invasive... The naturally occurring Verticillium nonalfalfae shows promise for biocontrol of the highly invasive Tree of Heaven (Ailan- thus altissima), but might also bear a risk for non-target tree species. In this study, we conducted inoculations on potted seed- lings of A. altissima as well as on eight indigenous and two invasive tree species associated with Tree of Heaven in Austria. Although vascular discolourations developed in all inoculated tree species, V. nonalfalfae was reisolated from Ailanthus and eight of the ten non-target-species, whereas typical disease symptoms and mortality only occurred on A. altissima. Results confirmed high susceptibility ( S) of A. altissima to V. nonalfalfae but indicated tolerance (T) of Acer campestre, Acer pseu- doplatanus and Quercus robur, possible resistance (PR) of Fraxinus excelsior, Populus nigra, Tilia cordata, Ulmus laevis and Ulmus minor and resistance (R) of Fraxinus pennsylvanica and Robinia pseudoacacia to this potential biocontrol agent. Results from seedling inoculations were confirmed by cursory field observations in Ailanthus-inoculated forest stands, where admixed A. campestre, A. pseudoplatanus, F. excelsior, Populus alba, R. pseudoacacia and U. laevis canopy trees remained asymptomatic, while mortality was induced in Ailanthus. Keywords Tree of Heaven · Ailanthus altissima · Verticillium nonalfalfae · Host range · Biological control · Non-target- effects Introduction Commission 2016). Furthermore, measures for early detec- tion, rapid eradication and/or management of these species According to the European Commission, the annually EU- have to be undertaken that also include the obligation of wide costs caused by invasive alien species (due to the costs landowners to actively control and eliminate these species. for health care, animal health, crop yield losses, damage to This is either a costly obligation or—based on the current infrastructure and the navigability of rivers, and damage to possibilities of a successful regulatory response—simply protected species) are estimated to sum up to EUR 12 billion impossible. Thus, the current list is a compromise and does per year (European Commission 2014). For that reason, the not comprise all ecologically relevant species, which are European Commission adopted a list that defines restric - undoubtedly defined as being invasive in Central Europe tions on keeping, importing, selling, breeding and growing (Essl and Rabitsch 2002, 2004; Kleinbauer et al. 2010; BfN of 37 invasive alien species of Union concern (European 2013; Info Flora 2014; LBV 2017). One of these unlisted species and a major woody pest in Austria is Ailanthus altissima (Mill.) Swingle 1916 (Sima- roubaceae)—also known as Tree of Heaven and henceforth Communicated by Gediminas Brazaitis. mentioned as Ailanthus. The tree was introduced into Aus- * E. Halmschlager tria in the late eighteenth century (Märter 1796; Borkhausen erhard.halmschlager@boku.ac.at 1803) and established rapidly as promenade, ornamental and O. Maschek shade tree during the nineteenth century (Neilreich 1846, oliver.maschek@boku.ac.at 1866; Wessely 1871). It was further cultivated as pulp wood (Härtel 1955; Schwarz 1955a, b), in windshields (Adamik Department of Forest and Soil Sciences, Institute of Forest Entomology, Forest Pathology and Forest Protection (IFFF), 1955), in welfare afforestations (Schimitschek 1952) but also University of Natural Resources and Life Sciences, Vienna as food source for honeybees (Gölles 1955). Widespread (BOKU), Peter-Jordan-Str. 82, 1190 Vienna, Austria Vol.:(0123456789) 1 3 198 European Journal of Forest Research (2018) 137:197–209 dissemination of Ailanthus was further promoted during the Subbarao 2014; Kasson et al. 2014, 2015). Thus, the full host twentieth century due to the colonization of sites which were range of V. nonalfalfae—especially for European species—is opened by bombs during World War II, as well as the colo- still unknown, because after the recent taxonomic changes in nization of derelict urban sites and railway areas, or fallow the genus Verticillium by Inderbitzin et al. (2011) all previ- land (Punz et al. 1998, 2004; Adler and Mrkvicka 2003). ous host records of Verticillium albo-atrum s.l. cannot be Ailanthus is a fast growing, highly invasive tree species transferred to V. nonalfalfae. They may refer to Verticillium with low requirements on soil quality and climate. Due to alfalfae, V. nonalfalfae or the distantly related V. albo-atrum its reproduction by root suckers, its fast and copious fructifi- s.s., which resemble each other in morphology. cation and its allelopathic properties (Hu 1979; Kowarik and Thus, the aim of this study was to investigate the effects Säumel 2007; Cáceres 2010; Wickert et al. 2017), Ailanthus of the Austrian V.  nonalfalfae-isolate G1/5 on the most alters natural tree species composition in riparian forests or common indigenous non-target, as well as on additional on dry gravel sites (Kowarik and Böcker 1984; Gutte et al. invasive tree species through artificial stem inoculations on 1987; Kowarik and Säumel 2007, 2008; Wickert et al. 2017). potted seedlings. The inoculation study was complemented It has therefore become a major problem in the warmer cli- by cursory field observations in Ailanthus-inoculated forest mate regions of Austria during the last 25 years. Besides stands with admixed Acer campestre, Acer pseudoplata- Ailanthus, there are also other invasive tree species such nus, Fraxinus excelsior, Populus alba, R. pseudoacacia and as box elder (Acer negundo), green ash (Fraxinus pennsyl- Ulmus laevis canopy trees. The results of the study provided vanica) and black locust (Robinia pseudoacacia) (Essl and important information on the host adaptation/specificity of Rabitsch 2002, 2004; Kleinbauer et al. 2010) which also the Austrian V.  nonalfalfae isolate and the potential risk invade open-canopy forests, riparian forests, dry gravel sites associated using this pathogen as biological control agent. or river banks in Austria. In order to repress or eliminate these invasive tree spe- cies, different mechanical, chemical or combined control Materials and methods methods have been considered for use (Ließ 2007; Probst 2012; Radtke et al. 2013; ÖBf 2014; Merceron et al. 2016). The stem inoculation experiment was conducted from May However, these methods are expensive, often not successful, 2013 to October 2014 on three-year-old potted seedlings an undesirable option (e.g. in near natural ecosystems or bio- of A. campestre, A.  pseudoplatanus, Ailanthus (control), sphere reserves) or even prohibited (e.g. in water protection F. excelsior, F.  pennsylvanica, Populus nigra, Quercus areas or in national parks). Thus, biological control of Ailan- robur, R. pseudoacacia, Tilia cordata, U. laevis and Ulmus thus and other invasive tree species using the wilt-inducing minor. Except for Ailanthus and F. pennsylvanica, seedlings fungus Verticillium nonalfalfae might become a promising were obtained from the tree nursery Schwanzer (Langen- alternative. In particular, because Acer, Ailanthus, Fraxinus schönbichl, Lower Austria) and the forest nursery Murauer and Robinia were listed in prior host lists as susceptible gen- (Hübing, Upper Austria). To cover a possible loss due to era to Verticillium (Spaulding 1958; Pegg and Brady 2002; transplantation, 35 bare-rooted seedlings of each species Sinclair and Lyon 2005 and Butin 2011). Verticillium spp. (80 –120 cm high) were purchased in April 2013 and trans- are native to Austria (Cech 1998; Maschek 2011; Maschek planted the next day into 5.5-l plastic pots filled with a ready and Halmschlager 2016a), and previous surveys and host mix potting soil for woody and perennial plants (Terra Vita range studies have confirmed pathogenicity and aggressive- Pflanzsubstrat, T6-Gehölze/Stauden; Franz Kranzinger ness and support host adaption of Verticillium nonalfalfae GmbH, Straßwalchen, Austria). All potted seedlings were to Ailanthus (Schall and Davis 2009; Kasson et al. 2014, kept in the garden of the institute and watered as neces- 2015; O’Neal and Davis 2015; Maschek and Halmschlager sary. In April 2015, seedlings were fertilized applying 20 g 2016a, 2017). Furthermore, V. nonalfalfae was suggested Osmocote Exact Hi.End 8-9 M (15-9-11+2MgO+TE) per to be pathogenic to R. pseudoacacia (Kasson et al. 2015; plastic pot. Kletzmayr 2016), whereas inoculations on box elder were Because seedlings of Ailanthus were not available from unsuccessful so far (Kasson et al. 2015). tree nurseries in 2013, rooted cuttings and excavated root However, the application of this wilt-inducing pathogen suckers, which were obtained from different trees from two in a forestal environment still bears the risk of non-target- sites (N48°17′22.29″ E16°20′47.249” and N48°20′21.959″ effects on associated tree species in Austria. Previous host E16°45′37.35″), were used for inoculation of Ailanthus. range studies referring to V. nonalfalfae had been carried out The same was true for F. pennsylvanica seedlings, which in North America yet and only five of the species defined were carefully excavated from a natural stand next to the as susceptible are indigenous (archaeophytes included) in river Danube (N48°19′5.783″ E16°20′26.66″). Cuttings of Europe (Acer platanoides, Humulus lupulus, Prunus avium, about 40 cm in length and about 1 cm in diameter and exca- Sambucus racemosa and Spinacia oleracea) (Inderbitzin and vated root suckers (80–150 cm in height) from Ailanthus, 1 3 European Journal of Forest Research (2018) 137:197–209 199 as well as excavated F. pennsylvanica seedlings measuring The stem inoculation experiment was repeated on 13 July 80–180 cm in height, were transplanted in the same substrate 2014 with 20 seedlings using the same inoculation method as the other seedlings and kept moist. Due to the physiologi- as in 2013, but control comprised only four seedlings for cal stress caused by cutting or excavation and in order to each species in 2014. In order to reduce partial girdling, provide enough time to establish roots and to ensure that seedlings were inoculated in 2014 at only one point at the excavated root suckers/seedlings were uninfected by other stem base approximately 7.5 cm above ground level, apply- pathogens, no inoculations were performed on Ailanthus and ing 1 × 1 ml conidial suspension. V. nonalfalfae-inoculated F. pennsylvanica in 2013. The same was true for Q. robur, seedlings comprised 15 randomly chosen seedlings, which which was heavily infested by Erysiphe alphitoides and were already treated in 2013 but did not develop any symp- has therefore been treated four times during the vegetation toms, and five so far untreated seedlings. Again, all seedlings period 2013 with a fungicide (wettable sulphur/COMPO were kept in the garden of the institute and watered as neces- BIO Mehltau-frei Thiovit Jet); thus, inoculation was post- sary. Since the rooted cuttings of Ailanthus, as well as the poned to 2014. On 23 August 2013, 20 healthy potted seed- F. pennsylvanica seedlings, developed well and appeared to lings of each tree species (except Ailanthus, F. pennsylvanica be vigorous and healthy in 2014, both species were included and Q. robur) were randomly selected and stem-inoculated in the stem inoculation experiment in 2014. The same was using the inoculation method described in Maschek and true for Q.  robur in 2014, on which Erysiphe infections Halmschlager (2016b) using a S tubai woodcarving tool could be kept to a minimum due to early fungicide treat- Sweep 7, size 6 mm (Art. No. 5507 06). If necessary, the ments. However, due to the mortality of seedlings caused inoculation slit was “closed” with the reverse side of the by the massive Erysiphe infection in the previous year, only gouge blade. Another six randomly chosen healthy seedlings 19 Q. robur seedlings could be subjected to V. nonalfalfae- of each tree species were treated with sterile water (using the stem inoculations in 2014. Again, development of disease same method and tool as for conidial stem inoculations) and symptoms was rated biweekly for all seedlings until the end served as controls. of the vegetation period applying the same adapted scale as Stem inoculations were performed using the V. nonal- described above. falfae-isolate G1/5 (GenBank Accession No. KT223526), At the termination of the experiment in November 2015, which was isolated from symptomatic Ailanthus in Bad all plants inoculated in 2014 were cut at ground level and Radkersburg, province of Styria. For production of fun- transported to the laboratory. Reisolations were made from gal inoculum, cultures of V. nonalfalfae grown on 2% malt three sections of the stem of each seedling: (1) from the extract agar [MEA; 20 g DiaMalt malt extract (Hefe Schweiz inoculation site, (2) 5 cm below the inoculation site and (3) AG, Stettfurt, Switzerland), 16 g Becoagar agar (W. Beh- 10 cm above the inoculation site (Table 2). For that purpose, rens & Co, Hamburg, Germany), 1000 ml tap water, 100 mg an approximately 4-cm-long sample was taken from each streptomycin sulphate (Calbiochem, Merck KGaA, Darm- site using sterile pruning shears. Samples were inspected stadt, Germany), added after autoclaving] and incubated at on both cross sections for the occurrence of vascular discol- 22–24 °C in the dark for 17 days were flooded with 20 ml ourations that are often associated with Verticillium infec- of sterile water. A glass spatula was used to release conidia tions (Sinclair and Lyon 2005; Butin 2011). Afterwards, from conidiophores and the resulting conidial suspension they were surface-sterilized using 96% ethanol for 1 min 7 −1 was adjusted to 1 × 10 spores ml . A germination test and then rinsed with sterile water for 30 s. Thereafter, each conducted immediately prior to inoculations ensured that sample was split longitudinally and tissue samples were rate of conidial germination exceeded 80%. Each seedling excised from vascular discolourations or—if available— was treated on two opposite points (one at the stem base from living sapwood tissue at the transition zone to necrotic approximately 5 cm above ground level and another on the tissue. Samples were placed (4 samples per Petri dish and opposite side of the stem approximately 10 cm above ground stem section) on 9-cm plastic Petri dishes containing 2% level) applying 2 × 0.5 ml conidial suspension or 2 × 0.5 ml MEA medium (as described above). Plates were sealed with sterile water for the controls. Following inoculation, all seed- Parafilm , labelled with a code for the tree species, tree lings were inspected biweekly for the development of dis- number and section code and incubated at 22–24 °C in the ease symptoms until the end of the vegetation period. Dis- dark for 12 days. Resulting isolates morphologically resem- ease severity was rated according to a slightly adapted scale bling V. nonalfalfae (Domsch et al. 1980; Inderbitzin et al. used by Bejarano-Alcázar et al. (1996) and Schall and Davis 2011) were subcultured on the same conditions for another (2009): 0 = no symptoms/healthy leaves, 1 = 1–33% foli- 18 days. Isolated fungal species other than V. nonalfalfae age affected/chlorotic leaves, 2 = 34–66% foliage affected/ were not further examined. A seedling was scored positive necrotic leaf margins, 3 = 67–99% foliage affected/wilting for V. nonalfalfae if the pathogen was reisolated from at least leaves, 4 = dead or defoliated seedling. one of the three sampled sections. 1 3 200 European Journal of Forest Research (2018) 137:197–209 Fig. 1 Discolourations in sap- wood of inoculated seedlings: a A. campestre; b A. pseudoplata- nus; c F. excelsior; d F. pennsyl- vanica; e P. nigra; f Q. robur; g R. pseudoacacia; h T. cordata; i U. laevis; j U. minor Photographs of vascular/sapwood discolourations were listed as susceptible to Verticillium spp. according to Engel- taken from the cross section and longitudinal section hard (1957), Himelick (1969), Pegg and Brady (2002) or (Fig. 1a–j) of representative samples of each species. If pos- Sinclair and Lyon (2005) (Table 1). However, observations sible, photographs were taken from the part opposite of the were not confirmed by a systematic survey on these sites. inoculation site, in order to avoid discolourations caused by embolism, following wound inoculation. In addition to the inoculation experiment on potted seed- Results lings, three sites within Ailanthus-inoculated or natural- infected forest stands were examined in summer 2015 for External disease symptoms and mortality wilting symptoms on non-target tree species: (1 + 2) two on seedlings sites in Lower Austria, where 20 Ailanthus trees had been artificially stem-inoculated in August 2011 and in June 2013, Stem inoculations on Ailanthus resulted in a rapid disease respectively, and (3) a forest plot in Gänserndorf (Lower progression on 100% (20 of 20 plants) of the trees in 2014: Austria) that became naturally infected with V. nonalfalfae The seedlings exhibited minor disease symptoms (mean in summer 2011 (Maschek and Halmschlager 2016a). Obser- disease severity [MDS] of 0.3) already 2 weeks post-inoc- vations focused on indigenous woody species, which were ulations (WPI), progressing rapidly reaching a MDS of 3.2 1 3 European Journal of Forest Research (2018) 137:197–209 201 Table 1 Site descriptions of mixed Ailanthus stands artificially inoculated or naturally infected by Verticillium nonalfalfae, as well as admixed tree species, all of which did not develop any disease symptoms Site Inoculation date Sea level Orientation/gradi- Soil type Stocking Composition of Stem Location, province ent level (in tree species (in number tenths) tenths) (N/ha) 1 30.08.2011 145 m Plain/0° Chernozem 0.9 0.7 Ailanthus 2.500 Niederweiden, (clayey to silty altissima Lower Austria to silty-clayey 0.1 Acer pseudo- suspended allu- platanus vial material). 0.1 Robinia pseu- At 30–100 cm doacacia gravel from river 0.1 other tree Danube species (Acer campestre, Sambucus nigra, Tilia cordata, Betula pendula) 2 04.06.2013 270 m South-east, slight Chernozem/Para- 0.4 0.3 Fagus sylvatica 2.000 Schützen am slope/10° rendzina middle- 0.2 Tilia cordata Gebirge, Burgen- to deep grounded 0.2 Ailanthus land altissima 0.2 Quercus cerris 0.1 Carpinus betulus 3 Natural 245 m Plain/0° Loess, mostly 1 0.4 Ailanthus 3.500 Hohenruppersdorf, infection in middle- to altissima Lower Austria 2010/2011 deep grounded 0.2 Fraxinus nutritious brown excelsior earths 0.2 Acer campestre 0.2 other tree species (Corylus avellana, Prunus serotina, Sorbus tormi- nalis) at the end of the vegetation period in 2014, resulting in 85% plants) developed chlorosis/necrosis 6 WPI and another (17 of 20 plants) dead Ailanthus trees by November 2015, 10% of the inoculated seedlings (2 of 20 plants) exhibited when the reisolations were carried out. 0% (0 of 4 plants) the same symptoms 8 WPI. The controls of F. pennsylvan- of the controls developed disease symptoms or died during ica remained asymptomatic and no seedling (inoculated or the evaluation period. control) died until the end of the evaluation period. Contrary to these results, stem-inoculated A. campestre, R. pseudoacacia inoculated in 2013 exhibited no symp- A. pseudoplatanus, U. laevis, U. minor, P. nigra, Q. robur toms until the end of the vegetation period. Regardless of and T. cordata as well as all controls developed no disease these results, 20% (4 of 20 plants) of the inoculated seed- symptoms throughout the observation period in 2013 or lings showed symptoms of dieback in spring/early sum- 2014. mer in the following year and were dead at the date of the No symptoms were also detected on stem-inoculated new inoculations in 2014 (13.07.2014). The same was true F. excelsior during the vegetation period in 2013, however, for 33% (2 of 6 plants) of the controls. However, dieback in 2014 20% (4 of 20 plants) of the inoculated seedlings also occurred on additionally reared untreated seedlings and 25% (1 of 4 plants) of the controls developed chloro- (i.e. seedlings that were neither inoculated with conidial sis, necrosis and curled leaves 6 WPI resulting in a MDS suspension nor with sterile water) in 2013 that served as of 0.13 for the inoculated seedlings and a MDS rating of reserves. Mortality reached 25% (2 of 8 plants) on these 0.25 for the controls at the end of the vegetation period untreated seedlings. in 2014. Regardless these symptoms, none of these seed- Seedlings of R. pseudoacacia inoculated in 2014 again lings died until the end of the evaluation period. Similar exhibited no symptoms until the end of the vegetation symptoms were developed on F. pennsylvanica, which was period but did not show any mortality in the following inoculated only in 2014: 15% of these seedlings (3 of 20 1 3 202 European Journal of Forest Research (2018) 137:197–209 Table 2 Results of the artificial inoculations of seedlings of non-target tree species carried out in 2013 and 2014 that were processed in Novem- ber 2015 Species Year of Seedlings Disease Mortality Vascular dis- Successful Successful reisola- Status inoculation inoculated/ symptoms (%) colourations reisolations tions from stem sec- controls (n) (%) (%)* total (%)* tions (%)* 2013 2014 2013 2014 2013 2014 2013 2014 Inoc. Control Inoc. Control − 5 cm 0 + 10 cm b b Ailanthus altissima – X – 20/4 – 100 – 85 100 0 66 0 66 0 0 S Acer campestre X X 20/6 20/4 0 0 0 0 30 0 85 0 40 50 65 T Acer pseudoplatanus X X 20/6 20/4 0 0 0 0 5 0 95 0 55 75 50 T Fraxinus excelsior X X 20/6 20/4 0 20 0 0 5 0 20 0 10 10 0 PR Fraxinus pennsylvanica – X – 20/4 – 25 – 0 15 0 0 0 0 0 0 R Populus nigra X X 20/6 20/4 0 0 0 0 5 0 10 0 5 5 0 PR Quercus robur – X – 19/4 – 0 – 0 42 0 63 0 21 42 26 T c c Robinia pseudoacacia X X 20/6 20/4 0 20 0 20 85 0 0 0 0 0 0 R Tilia cordata X X 20/6 20/4 0 0 0 0 60 0 25 0 5 15 10 PR Ulmus laevis X X 20/6 20/4 0 0 0 0 100 0 10 0 5 5 0 PR Ulmus minor X X 20/6 20/4 0 0 0 0 60 0 5 0 0 0 5 PR S = susceptible (wilt/mortality, high rate of reisolation), T = tolerant (no wilt/mortality, high rate of reisolation), PR = possibly resistant (no wilt/mortality, low rate of reisolation), R = resistant (no wilt/mortality, no reisolation) Data of columns with headers indicated by a single asterisk (*) refer to seedling inoculations in 2014 Reisolations were carried out on three different sections of the stem: from the inoculation site (0 cm), 5 cm below the inoculation site (− 5 cm) and 10 cm above the inoculation site (+ 10 cm) Reisolations were carried out only on seedlings that still exhibited at least some living stem/root tissue Mortality attributed to other abiotic/biotic reasons spring/early summer. No symptoms were observed on the controls in 2013 and 2014. Vascular discolourations on seedlings Vascular discolourations in the sapwood varied in colour, appearance and in intensity among the inoculated tree spe- cies (Fig. 1a–j). Furthermore, there was also a high varia- tion in the number of seedlings that exhibited such vascu- lar discolourations due to artificial infections between the tested species. In Ailanthus, vascular discolourations were yellowish/orange. However, these symptoms could only be observed on seedlings (3 of 3 plants) that still exhibited at least some living cortex chlorenchyma (Table  2). In con- trast, dead Ailanthus seedlings (85% or 17 of 20 plants) were lacking these typical yellowish/orange discolourations but exhibited brownish, necrotic cortex chlorenchyma (Fig. 2) and sapwood and were additionally characterized by a foul smell. Orange/brownish discolourations within the sapwood were typically for F. excelsior, F. pennsylvanica and P. nigra (Fig. 1c–e). However, in contrast to Ailanthus, discoloura- tions occurred only on 5% (1 of 20 plants) on F. excelsior and P. nigra and on 15% (3 of 20 plants) of the inoculated seedlings of F. pennsylvanica (Table 2). Brownish discol- ourations of the sapwood (Fig. 1f, h) were found on 42% (8 Fig. 2 Brownish, necrotic cortex chlorenchyma and necrotic sapwood of 19 plants) of the inoculated seedlings of Q. robur and on of dead Ailanthus seedlings (red arrows) observed during examina- 60% (12 of 20 plants) of the examined T. cordata-seedlings tions in November 2015 1 3 European Journal of Forest Research (2018) 137:197–209 203 (Table 2). A. campestre and U. minor exhibited brownish/ sections (14.1% at − 5 cm and 15.6% at + 10 cm from the blackish discolourations that were found on 30% (6 of 20 point of inoculation). On only two tree species, reisolations plants) and on 60% (12 of 20 plants), respectively (Table 2; were more successful on a stem section, which was not Fig. 1a, j). The most distinctive vascular symptoms were the the point of inoculation: on A. campestre (65% or 13 of 20 greenish/blackish discolourations found on A. pseudoplata- plants) and U. minor (5% or 1 of 20 plants), both at + 10 cm. nus, R. pseudoacacia and U. laevis (Fig.  1b, g, i). While Field observations in mature stands subjected to inocula- these symptoms were quite rare on A. pseudoplatanus (5% tions/natural infection of Ailanthus canopy trees on three or 1 of 20 plants), they occurred quite commonly on R. pseu- forest sites revealed that there was no formation of wilt- doacacia (85% or 17 of 20 plants) and were consistently ing symptoms on admixed non-target tree species such found on inoculated seedlings of U. laevis (100% or 20 of 20 as A. campestre, A. pseudoplatanus, F. excelsior, P. alba, plants) (Table 2). In contrast, none of the controls exhibited R. pseudoacacia and U. laevis. However, no isolations were vascular discolourations, which could be related to Verticil- carried out from that symptomless admixed tree species. In lium infections (Table 2). contrast, severe wilting symptoms, dieback and mortality were observed on inoculated/naturally infected Ailanthus Reisolations of V. nonalfalfae from seedlings trees as well as on adjacent Ailanthus trees that got infected via roots grafts (Fig. 4). Successful rate of reisolation differed considerably between the tested tree species. It ranged from 95% in A. pseudopla- tanus to 0% in F. pennsylvanica and R. pseudoacacia. Fur- thermore, results revealed that rate of reisolation did not cor- Discussion relate with the intensity of vascular discolourations. Thus, there were species with heavily discoloured sapwood but low Knowing the potential host range of V. nonalfalfae is of great rate of successful reisolation (R. pseudoacacia, U. laevis), importance for a hazard and exposure risk assessment. It but there were also species of which V. nonalfalfae could be is also an inevitable prerequisite to avoid undesirable non- reisolated quite common although vascular discolourations target-effects on associated tree species using that fungus were rare (A. pseudoplatanus) as well as species that were for the biological control of Ailanthus or other invasive tree characterized by both, a low rate of successful reisolation species. and a low rate of seedlings exhibiting vascular discoloura- However, the descriptions of V. nonalfalfae and the mor- tions (Table 2). phologically indistinguishable V. alfalfae, both resembling Due to the fact that 17 of 20 seedlings of Ailanthus were the distantly related V. albo-atrum in morphology, were pro- already dead at the time of processing, V. nonalfalfae could vided just a few years ago (Inderbitzin et al. 2011). Thus, be reisolated from 66% (2 of 3) of the seedlings that still previous host range studies of V. nonalfalfae are rare and exhibited at least some living stem or root tissue but showed mainly focus on agricultural crops (Inderbitzin and Subbarao an advanced stage of dieback and subsequent colonization 2014), ornamentals (Garibaldi et al. 2016) or woody species by saprobionts. V. nonalfalfae could not be reisolated from native to North America (Schall and Davis 2009; Kasson controls of Ailanthus. et al. 2014, 2015). In Europe, V. nonalfalfae was confirmed Besides Ailanthus, reisolations were also successful on for the first time just recently from a woody host (Maschek another 8 of 11 tree species (Table  2): Reisolations were and Halmschlager 2016a). Except for A. platanoides, Prunus most successful on A.  pseudoplatanus (95% or 19 of 20 avium and Sambucus racemosa, host range studies on woody plants) although vascular discolourations were found on plants native to Europe are therefore completely lacking so only 5% of these seedlings, followed by A. campestre with far. 85% (17 of 20 plants) and Q.  robur with 63% (12 of 19 Although a long list of diverse hosts—including also plants). V. nonalfalfae could also be successfully reisolated many tree species—is known for V. albo-atrum s.l. (Pegg from 25% (5 of 20 plants) of the seedlings of T.  cordata and Brady 2002; Sinclair and Lyon 2005; Butin 2011), it is and from 20% (4 of 20 plants) of F. excelsior. Low rates of not possible to relate the host range information from previ- successful reisolations were obtained for P. nigra, U. laevis ous literature on V. albo-atrum s.l. to the newly delimitated (10% or 2 of 20 plants) and U. minor (5% or 1 of 20 plants). Verticillium-species V. albo-atrum s.s., V. alfalfae or V. non- V. nonalfalfae could not be reisolated at all from F. penn- alfalfae (Inderbitzin et al. 2011), as long as molecular data or sylvanica and R. pseudoacacia, although each species was detailed morphological descriptions or cultures are lacking. sampled 240 times. In order to expand the information regarding host range of Reisolations were most successful at the point of inocu- V. nonalfalfae, we tested the susceptibility of eight indig- lation (overall rate of reisolation: 20%), and there was only enous non-target tree species, often associated with Tree of a minor difference between the two other sampled stem Heaven, and two invasive exotic tree species. 1 3 204 European Journal of Forest Research (2018) 137:197–209 In our study, V. nonalfalfae induced characteristic wilt- Rudolph 1931; Carter 1938; Weiss 1940; Engelhard 1957; ing symptoms and high mortality only on the target host Himelick 1969; Pegg and Brady 2002), referring to the for- Ailanthus, whereas limited or no impact was detected on mer V. albo-atrum s.l., also A. campestre, A. pseudoplata- non-target tree species, thus indicating a high degree of nus, as well as other Acer species, were considered suscep- host specificity. In addition, consistent vascular discolora- tible. Thus, differences concerning susceptibility/tolerance tions and the successful reisolation of the applied isolate of woody host species between these prior and recent studies indicate the high susceptibility (S) of Ailanthus seedlings to indicate a narrower host range of V. nonalfalfae compared V. nonalfalfae. Similar results had been obtained by Schall to V. albo-atrum s.l. and Davis (2009) and Kasson et al. (2015), testing the host In contrast to Q. robur, which was considered tolerant range for V. nonalfalfae in the eastern USA. Wilt, dieback in our study, V.  nonalfalfae could not be reisolated from and mortality were also observed on artificially inoculated inoculated northern red oak (Quercus rubra) and chestnut canopy trees of Ailanthus on several sites in eastern Aus- oak (Quercus montana) and has therefore been classified tria (Maschek 2011; Maschek and Halmschlager 2017). as resistant by Schall and Davis (2009) and Kasson et al. The same symptoms were also observed on Ailanthus trees (2015). According to field observations in Ailanthus-inoc- adjacent to the inoculated trees, all of which were considered ulated forest stands, resistance was also suggested by the to be connected via root grafts. Intraspecific transmission latter authors for black oak (Quercus velutina). However, by root crafts has already been demonstrated by O’Neal and no inoculation studies had been performed on this tree spe- Davis (2015). Extensive dieback and mortality of Ailanthus cies. In addition, vascular discolourations were observed canopy trees additionally confirm the high virulence of the twice as much in Q. robur (8 of 19 seedlings) in our study, European V. nonalfalfae-isolate G1/5 (Fig. 4) found in seed- compared to Q. rubra (2 of 10 trees) in the study carried ling inoculations and qualify this pathogen as potential bio- out by Kasson et al. (2015). According to previous papers logical control agent for this invasive species (Maschek and dealing with V. albo-atrum s.l., the genus Quercus includes Halmschlager 2016a). both susceptible and resistant species (Himelick 1969; Pegg In contrast to the high susceptibility of Ailanthus, V. non- and Brady 2002; Sinclair and Lyon 2005). V. albo-atrum s.l. alfalfae had no or only limited impact on the tested non- has so far been recorded on the surface of Q. robur acorns target and the two invasive tree species in this study: Based (Urosevic 1987), but is not mentioned in the detailed host on disease symptoms, secondary symptoms like vascular index of V. albo-atrum s.l. published by Engelhard (1957). discolourations and the rate of successful reisolations, tested In F.  excelsior, P.  nigra, T.  cordata, U.  laevis and tree species were assigned as being tolerant, possibly resist- U.  minor, rate of reisolations was rather low and did not ant or resistant. exceed 15% for each stem section (Table 2). Because these Inoculated A. campestre, A. pseudoplatanus and Q. robur species also did not develop disease symptoms (except for exhibited neither primary disease symptoms (chloroses, F. excelsior) and exhibited no mortality, all five species are necroses or wilt) nor mortality; however, V. nonalfalfae was considered possibly resistant (PR). Disease symptoms on frequently reisolated from inoculated seedlings. Recovery the inoculated seedlings of F. excelsior developed simulta- of the pathogen from these asymptomatic seedlings indi- neously to the same extent on the controls and comprised cates a low level of systemic infection that did not result chlorotic and necrotic leaves with typical swellings on the in symptom development. Thus, these three species were rachises. Visual inspections indicated that these swellings considered tolerant (T) to European V. nonalfalfae-isolate could be attributed to the gall-forming midge Dasineura G1/5 (Table 2). Irrespectively of the high rate of reisolations, fraxini; thus, displayed symptoms were not related to V. non- formation of vascular discolourations was only observed to alfalfae infections. a considerably lesser extent on the examined samples. Simi- Similar results were obtained by Schall and Davis (2009) lar results concerning tolerance to V. nonalfalfae had been for V. nonalfalfae-inoculated seedlings and canopy trees of obtained in a host range study by Kasson et al. (2015) for red white ash (Fraxinus americana), which was considered as maple (Acer rubrum) and sugar maple (Acer saccharum), tolerant (= resistant according to our classification), because but also for Korean evodia (Tetradium daniellii) and Tree of reisolation was unsuccessful in all cases. Susceptibility of Paradise (Simarouba glauca). In contrast, inoculated striped F. excelsior to Verticillium wilt (Sinclair and Lyon 2005) maple (Acer pennsylvanicum) reached similar disease levels was usually attributed to V. dahliae infections in previous as Ailanthus in a study carried out in eastern USA by Schall reports (Hiemstra 1995; Heffer and Regan 1996; Schuring and Davis (2009) and was therefore considered susceptible and van der Schaaf 1999). However, Himelick (1969) also to V.  nonalfalfae (isolate PSU140). Susceptibility to this recovered V. albo-atrum s.l. from branch specimens of black, V.  nonalfalfae isolate was also demonstrated for Norway blue, European, green and white ash and was able to reiso- maple (A. platanoides) and Japanese maple (Acer japonica) late the fungus from all inoculated seedlings except black by Kasson et al. (2015). In prior host lists (compiled by ash, which was not included in his pathogenicity test. Green 1 3 European Journal of Forest Research (2018) 137:197–209 205 ash was also included in the host list of V. albo-atrum s.l. compiled by Engelhard (1957). Among poplar species, susceptibility to V. nonalfalfae was previously only tested for White poplar (P. alba) and Bigtooth aspen (Populus grandidentata) (Kasson et  al. 2015). However, the authors did not provide any results for the two species. Assuming that they did not develop symp- toms and were resistant, these results would be similar to those in our study, in which reisolations and vascular discol- ourations (Fig. 1e) were obtained for P. nigra, but occurred very rarely. In contrast to the results related to V. nonalfalfae, the host range of V. albo-atrum s.l. comprised susceptible Populus species such as Populus tremula (Liese 1933; Sin- clair and Lyon 2005). T. cordata exhibited neither wilt symptoms nor mortality (Table 2), which is similar to the results obtained by Kasson et al. (2015) for Tilia americana—this species was therefore considered resistant. In contrast to this study, V. nonalfal- fae was recovered from a quarter of all inoculated seed- lings. Vascular discolourations were found to a lower extent (60%, Fig. 1h) on T. cordata when compared to the results of T. americana (93%, Kasson et al. 2015). According to Fig. 3 Unspecific chloroses and necroses on F.  pennsylvanica in early September 2014 Sinclair and Lyon (2005), the genus Tilia includes both sus- ceptible and resistant species. In prior papers (Wollenweber 1929) and host lists compiled by Engelhard (1957), Himelick according to Heffer and Regan (1996), Hiemstra and Harris (1998) and Sinclair and Lyon (2005), Verticillium wilt of (1969) and Pegg and Brady (2002), T. cordata is frequently listed as susceptible host for V. albo-atrum s.l. along with F. pennsylvanica is generally associated with wilting leaves that remain greenish or is characterized by necroses; thus, other Tilia-species from Europe and Northern America. Both inoculated Ulmus species displayed neither wilt symptoms were not specific for Verticillium wilt. As there were no typical swellings on the leaf rachises, symptoms on symptoms nor mortality (Table  2), but regardless of the absence of these exterior symptoms, vascular discoloura- F. pennsylvanica could also not be attributed to infestation by D. fraxini, as it was the case in F. excelsior. In fact, symp- tions were commonly found in seedlings of U. laevis (100%, Fig. 1i) and U. minor (60%, Fig. 1j). Nevertheless, V. non- toms more resembled to manganese or magnesium deficien- cies (Sinclair and Lyon 2005; Hartmann et al. 2007; Butin alfalfae was reisolated only rarely from those vascular dis- colourations, indicating that the fungus very likely induced et al. 2010), but since nutritional status of seedlings was not further analysed, symptoms might alternatively be attributed these secondary disease symptoms, but could barely sur- vive in both Ulmus species until reisolations in Nov. 2015 to other biotic or abiotic agents. Regardless of these unspe- cific external symptoms, F.  pennsylvanica also developed (i.e. 14 months post-inoculation). Very similar results were obtained by Kasson et al. (2015) for Ulmus americana and less pronounced orange/brownish vascular discolourations (Fig. 1d). However, V. nonalfalfae could not be reisolated Ulmus  pumila: (1) Both species developed neither wilt symptoms nor mortality, (2) both Ulmus species exhibited from any of those discolourations; thus, F. pennsylvanica was considered resistant (R) to V. nonalfalfae. In contrast, vascular discolourations (U. americana 100%, U. pumila 40%) and (3) V. nonalfalfae could not be reisolated from the genus Fraxinus is listed as susceptible to Verticillium spp. (Himelick 1969; Heffer and Regan 1996; Sinclair and any elm-tree. However, U. americana, Ulmus glabra (Syn.: U. montana), U. minor (Syn.: U. campestris), Ulmus parvi- Lyon 2005; Butin et al. 2010) in older publications, taking not into account the recent taxonomic changes (Inderbitzin folia, Ulmus  procera and Ulmus  rubra are mentioned as being susceptible to V. albo-atrum s.l. in the host lists com- et al. 2011). Symptoms on R. pseudoacacia already developed in early piled by Rudolph (1931), Carter (1938), Engelhard (1957), Himelick (1969) or Pegg and Brady (2002). summer 2014 (before the 2014-inoculation was conducted) and occurred not only on Verticillium-inoculated seedlings, Like in F. excelsior, some F. pennsylvanica seedlings also developed chlorotic and necrotic leaves (Fig. 3). However, but also on the controls as well as on seedlings that were 1 3 206 European Journal of Forest Research (2018) 137:197–209 Fig. 4 Canopy trees in an Ailanthus-inoculated mixed forest stand A. pseudoplatanus, F. excelsior (red arrow), P. alba, R. pseudoacacia, in late August 2015, four years after inoculations; Ailanthus trees U. laevis remained asymptomatic showed mortality (blue arrows), whereas admixed A.  campestre, kept in 2013 in reserve. Thus, symptoms cannot be related are in contrast to prior publications and host lists referring to Verticillium infections but were rather related to drought. to V. albo-atrum s.l. (Goidanich 1935; Carter 1938; Engel- Although vascular discolourations were found to the second hard 1957; Himelick 1969; Pegg and Brady 2002; Sinclair most frequency on R. pseudoacacia, V. nonalfalfae could not and Lyon 2005) in which R.  pseudoacacia is considered be reisolated from any of the inoculated seedlings (Table 2). susceptible. Thus, this tree species was also categorized as resistant to Overall, results regarding vascular discolourations and the Austrian V.  nonalfalfae strain. In contrast, artificial the rate of successful reisolations obtained in our seedling inoculations with V. nonalfalfae conducted by Kasson et al. inoculation experiment possibly overestimate the impact of (2015) in 2009 and 2010 induced wilt on 20 and 80% of the V.  nonalfalfae under field conditions, because a success- inoculated plants, but resulted in only 0 and 10% mortality, ful natural infection by this soilborne pathogen might be respectively. Similar to our results, vascular discolourations counteracted or even prevented by root defences (Blanchette were consistently observed on trees inoculated in 2010, and Biggs 1992), which were bypassed by our stem inocula- whereas no discolourations were detected in trees inocu- tion method (Maschek and Halmschlager 2016b). There are lated in 2009 (Kasson et al. 2015). However, sample size also various effects influencing a natural infection or the in 2009 comprised only five black locust trees. Our results establishment of the pathogen within the host: (1) protec- tive effects of mycorrhizal fungi against Verticillium infec- tions (Karagiannidis et al. 2002), (2) synergistic effects of Symptoms only occurred on seedlings that had been moved—with- out the knowledge of the authors—to an unfavourable sun-exposed root-knot nematodes and Verticillium (Santamour 1992) or area of the institute’s garden in order to provide space for another (3) antagonistic effects of xylem-colonizing bacteria (Hall experiment. All other R. pseudoacacia seedlings remained symptom- et al. 1986). free. Seedlings subjected to drought did not recover until the end of the vegetation period 2014 and were classified as loss. 1 3 European Journal of Forest Research (2018) 137:197–209 207 Open Access This article is distributed under the terms of the Crea- Additionally, the applied spore concentration for sure tive Commons Attribution 4.0 International License (http://creat iveco exceeds the amount of conidia found in natural infections mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- (Pegg and Brady 2002). Thus, results indicating suscepti- tion, and reproduction in any medium, provided you give appropriate bility (which was only the case for Ailanthus in our study) credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. in artificial inoculations have to be treated with caution, whereas the absence of wilting symptoms may anyway indicate tolerance or true resistance. Nevertheless, the for- mation of vascular discolourations confirmed the success- References ful transmission of inoculum into the xylem, resulting in defence reactions such as occlusion of vessels (Tippett and Adamik K (1955) Der Götterbaum als Faserholz. Centbl gesamte Forst- Shigo 1981; Kasson et al. 2015; Maschek and Halmschlager wes 74:85–94 2016b). Adler W, Mrkvicka AC (2003) Die Flora Wiens gestern und heute. Results of seedling inoculations are supported for five (A. Verlag des Naturhistorischen Museums Wien, Wien Bejarano-Alcázar J, Blanco-López MA, Melero JM, Jiménez-Díaz RM campestre, A. pseudoplatanus, F. excelsior, R. pseudoacacia (1996) Etiology, importance, and distribution of Verticillium wilt and U. laevis) out of the tested species by field observations of cotton in Southern Spain. 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Effects of Verticillium nonalfalfae on Ailanthus altissima and associated indigenous and invasive tree species in eastern Austria

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Springer Journals
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Copyright © 2018 by The Author(s)
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Life Sciences; Forestry; Plant Sciences; Plant Ecology
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1612-4669
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1612-4677
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10.1007/s10342-018-1099-y
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Abstract

The naturally occurring Verticillium nonalfalfae shows promise for biocontrol of the highly invasive Tree of Heaven (Ailan- thus altissima), but might also bear a risk for non-target tree species. In this study, we conducted inoculations on potted seed- lings of A. altissima as well as on eight indigenous and two invasive tree species associated with Tree of Heaven in Austria. Although vascular discolourations developed in all inoculated tree species, V. nonalfalfae was reisolated from Ailanthus and eight of the ten non-target-species, whereas typical disease symptoms and mortality only occurred on A. altissima. Results confirmed high susceptibility ( S) of A. altissima to V. nonalfalfae but indicated tolerance (T) of Acer campestre, Acer pseu- doplatanus and Quercus robur, possible resistance (PR) of Fraxinus excelsior, Populus nigra, Tilia cordata, Ulmus laevis and Ulmus minor and resistance (R) of Fraxinus pennsylvanica and Robinia pseudoacacia to this potential biocontrol agent. Results from seedling inoculations were confirmed by cursory field observations in Ailanthus-inoculated forest stands, where admixed A. campestre, A. pseudoplatanus, F. excelsior, Populus alba, R. pseudoacacia and U. laevis canopy trees remained asymptomatic, while mortality was induced in Ailanthus. Keywords Tree of Heaven · Ailanthus altissima · Verticillium nonalfalfae · Host range · Biological control · Non-target- effects Introduction Commission 2016). Furthermore, measures for early detec- tion, rapid eradication and/or management of these species According to the European Commission, the annually EU- have to be undertaken that also include the obligation of wide costs caused by invasive alien species (due to the costs landowners to actively control and eliminate these species. for health care, animal health, crop yield losses, damage to This is either a costly obligation or—based on the current infrastructure and the navigability of rivers, and damage to possibilities of a successful regulatory response—simply protected species) are estimated to sum up to EUR 12 billion impossible. Thus, the current list is a compromise and does per year (European Commission 2014). For that reason, the not comprise all ecologically relevant species, which are European Commission adopted a list that defines restric - undoubtedly defined as being invasive in Central Europe tions on keeping, importing, selling, breeding and growing (Essl and Rabitsch 2002, 2004; Kleinbauer et al. 2010; BfN of 37 invasive alien species of Union concern (European 2013; Info Flora 2014; LBV 2017). One of these unlisted species and a major woody pest in Austria is Ailanthus altissima (Mill.) Swingle 1916 (Sima- roubaceae)—also known as Tree of Heaven and henceforth Communicated by Gediminas Brazaitis. mentioned as Ailanthus. The tree was introduced into Aus- * E. Halmschlager tria in the late eighteenth century (Märter 1796; Borkhausen erhard.halmschlager@boku.ac.at 1803) and established rapidly as promenade, ornamental and O. Maschek shade tree during the nineteenth century (Neilreich 1846, oliver.maschek@boku.ac.at 1866; Wessely 1871). It was further cultivated as pulp wood (Härtel 1955; Schwarz 1955a, b), in windshields (Adamik Department of Forest and Soil Sciences, Institute of Forest Entomology, Forest Pathology and Forest Protection (IFFF), 1955), in welfare afforestations (Schimitschek 1952) but also University of Natural Resources and Life Sciences, Vienna as food source for honeybees (Gölles 1955). Widespread (BOKU), Peter-Jordan-Str. 82, 1190 Vienna, Austria Vol.:(0123456789) 1 3 198 European Journal of Forest Research (2018) 137:197–209 dissemination of Ailanthus was further promoted during the Subbarao 2014; Kasson et al. 2014, 2015). Thus, the full host twentieth century due to the colonization of sites which were range of V. nonalfalfae—especially for European species—is opened by bombs during World War II, as well as the colo- still unknown, because after the recent taxonomic changes in nization of derelict urban sites and railway areas, or fallow the genus Verticillium by Inderbitzin et al. (2011) all previ- land (Punz et al. 1998, 2004; Adler and Mrkvicka 2003). ous host records of Verticillium albo-atrum s.l. cannot be Ailanthus is a fast growing, highly invasive tree species transferred to V. nonalfalfae. They may refer to Verticillium with low requirements on soil quality and climate. Due to alfalfae, V. nonalfalfae or the distantly related V. albo-atrum its reproduction by root suckers, its fast and copious fructifi- s.s., which resemble each other in morphology. cation and its allelopathic properties (Hu 1979; Kowarik and Thus, the aim of this study was to investigate the effects Säumel 2007; Cáceres 2010; Wickert et al. 2017), Ailanthus of the Austrian V.  nonalfalfae-isolate G1/5 on the most alters natural tree species composition in riparian forests or common indigenous non-target, as well as on additional on dry gravel sites (Kowarik and Böcker 1984; Gutte et al. invasive tree species through artificial stem inoculations on 1987; Kowarik and Säumel 2007, 2008; Wickert et al. 2017). potted seedlings. The inoculation study was complemented It has therefore become a major problem in the warmer cli- by cursory field observations in Ailanthus-inoculated forest mate regions of Austria during the last 25 years. Besides stands with admixed Acer campestre, Acer pseudoplata- Ailanthus, there are also other invasive tree species such nus, Fraxinus excelsior, Populus alba, R. pseudoacacia and as box elder (Acer negundo), green ash (Fraxinus pennsyl- Ulmus laevis canopy trees. The results of the study provided vanica) and black locust (Robinia pseudoacacia) (Essl and important information on the host adaptation/specificity of Rabitsch 2002, 2004; Kleinbauer et al. 2010) which also the Austrian V.  nonalfalfae isolate and the potential risk invade open-canopy forests, riparian forests, dry gravel sites associated using this pathogen as biological control agent. or river banks in Austria. In order to repress or eliminate these invasive tree spe- cies, different mechanical, chemical or combined control Materials and methods methods have been considered for use (Ließ 2007; Probst 2012; Radtke et al. 2013; ÖBf 2014; Merceron et al. 2016). The stem inoculation experiment was conducted from May However, these methods are expensive, often not successful, 2013 to October 2014 on three-year-old potted seedlings an undesirable option (e.g. in near natural ecosystems or bio- of A. campestre, A.  pseudoplatanus, Ailanthus (control), sphere reserves) or even prohibited (e.g. in water protection F. excelsior, F.  pennsylvanica, Populus nigra, Quercus areas or in national parks). Thus, biological control of Ailan- robur, R. pseudoacacia, Tilia cordata, U. laevis and Ulmus thus and other invasive tree species using the wilt-inducing minor. Except for Ailanthus and F. pennsylvanica, seedlings fungus Verticillium nonalfalfae might become a promising were obtained from the tree nursery Schwanzer (Langen- alternative. In particular, because Acer, Ailanthus, Fraxinus schönbichl, Lower Austria) and the forest nursery Murauer and Robinia were listed in prior host lists as susceptible gen- (Hübing, Upper Austria). To cover a possible loss due to era to Verticillium (Spaulding 1958; Pegg and Brady 2002; transplantation, 35 bare-rooted seedlings of each species Sinclair and Lyon 2005 and Butin 2011). Verticillium spp. (80 –120 cm high) were purchased in April 2013 and trans- are native to Austria (Cech 1998; Maschek 2011; Maschek planted the next day into 5.5-l plastic pots filled with a ready and Halmschlager 2016a), and previous surveys and host mix potting soil for woody and perennial plants (Terra Vita range studies have confirmed pathogenicity and aggressive- Pflanzsubstrat, T6-Gehölze/Stauden; Franz Kranzinger ness and support host adaption of Verticillium nonalfalfae GmbH, Straßwalchen, Austria). All potted seedlings were to Ailanthus (Schall and Davis 2009; Kasson et al. 2014, kept in the garden of the institute and watered as neces- 2015; O’Neal and Davis 2015; Maschek and Halmschlager sary. In April 2015, seedlings were fertilized applying 20 g 2016a, 2017). Furthermore, V. nonalfalfae was suggested Osmocote Exact Hi.End 8-9 M (15-9-11+2MgO+TE) per to be pathogenic to R. pseudoacacia (Kasson et al. 2015; plastic pot. Kletzmayr 2016), whereas inoculations on box elder were Because seedlings of Ailanthus were not available from unsuccessful so far (Kasson et al. 2015). tree nurseries in 2013, rooted cuttings and excavated root However, the application of this wilt-inducing pathogen suckers, which were obtained from different trees from two in a forestal environment still bears the risk of non-target- sites (N48°17′22.29″ E16°20′47.249” and N48°20′21.959″ effects on associated tree species in Austria. Previous host E16°45′37.35″), were used for inoculation of Ailanthus. range studies referring to V. nonalfalfae had been carried out The same was true for F. pennsylvanica seedlings, which in North America yet and only five of the species defined were carefully excavated from a natural stand next to the as susceptible are indigenous (archaeophytes included) in river Danube (N48°19′5.783″ E16°20′26.66″). Cuttings of Europe (Acer platanoides, Humulus lupulus, Prunus avium, about 40 cm in length and about 1 cm in diameter and exca- Sambucus racemosa and Spinacia oleracea) (Inderbitzin and vated root suckers (80–150 cm in height) from Ailanthus, 1 3 European Journal of Forest Research (2018) 137:197–209 199 as well as excavated F. pennsylvanica seedlings measuring The stem inoculation experiment was repeated on 13 July 80–180 cm in height, were transplanted in the same substrate 2014 with 20 seedlings using the same inoculation method as the other seedlings and kept moist. Due to the physiologi- as in 2013, but control comprised only four seedlings for cal stress caused by cutting or excavation and in order to each species in 2014. In order to reduce partial girdling, provide enough time to establish roots and to ensure that seedlings were inoculated in 2014 at only one point at the excavated root suckers/seedlings were uninfected by other stem base approximately 7.5 cm above ground level, apply- pathogens, no inoculations were performed on Ailanthus and ing 1 × 1 ml conidial suspension. V. nonalfalfae-inoculated F. pennsylvanica in 2013. The same was true for Q. robur, seedlings comprised 15 randomly chosen seedlings, which which was heavily infested by Erysiphe alphitoides and were already treated in 2013 but did not develop any symp- has therefore been treated four times during the vegetation toms, and five so far untreated seedlings. Again, all seedlings period 2013 with a fungicide (wettable sulphur/COMPO were kept in the garden of the institute and watered as neces- BIO Mehltau-frei Thiovit Jet); thus, inoculation was post- sary. Since the rooted cuttings of Ailanthus, as well as the poned to 2014. On 23 August 2013, 20 healthy potted seed- F. pennsylvanica seedlings, developed well and appeared to lings of each tree species (except Ailanthus, F. pennsylvanica be vigorous and healthy in 2014, both species were included and Q. robur) were randomly selected and stem-inoculated in the stem inoculation experiment in 2014. The same was using the inoculation method described in Maschek and true for Q.  robur in 2014, on which Erysiphe infections Halmschlager (2016b) using a S tubai woodcarving tool could be kept to a minimum due to early fungicide treat- Sweep 7, size 6 mm (Art. No. 5507 06). If necessary, the ments. However, due to the mortality of seedlings caused inoculation slit was “closed” with the reverse side of the by the massive Erysiphe infection in the previous year, only gouge blade. Another six randomly chosen healthy seedlings 19 Q. robur seedlings could be subjected to V. nonalfalfae- of each tree species were treated with sterile water (using the stem inoculations in 2014. Again, development of disease same method and tool as for conidial stem inoculations) and symptoms was rated biweekly for all seedlings until the end served as controls. of the vegetation period applying the same adapted scale as Stem inoculations were performed using the V. nonal- described above. falfae-isolate G1/5 (GenBank Accession No. KT223526), At the termination of the experiment in November 2015, which was isolated from symptomatic Ailanthus in Bad all plants inoculated in 2014 were cut at ground level and Radkersburg, province of Styria. For production of fun- transported to the laboratory. Reisolations were made from gal inoculum, cultures of V. nonalfalfae grown on 2% malt three sections of the stem of each seedling: (1) from the extract agar [MEA; 20 g DiaMalt malt extract (Hefe Schweiz inoculation site, (2) 5 cm below the inoculation site and (3) AG, Stettfurt, Switzerland), 16 g Becoagar agar (W. Beh- 10 cm above the inoculation site (Table 2). For that purpose, rens & Co, Hamburg, Germany), 1000 ml tap water, 100 mg an approximately 4-cm-long sample was taken from each streptomycin sulphate (Calbiochem, Merck KGaA, Darm- site using sterile pruning shears. Samples were inspected stadt, Germany), added after autoclaving] and incubated at on both cross sections for the occurrence of vascular discol- 22–24 °C in the dark for 17 days were flooded with 20 ml ourations that are often associated with Verticillium infec- of sterile water. A glass spatula was used to release conidia tions (Sinclair and Lyon 2005; Butin 2011). Afterwards, from conidiophores and the resulting conidial suspension they were surface-sterilized using 96% ethanol for 1 min 7 −1 was adjusted to 1 × 10 spores ml . A germination test and then rinsed with sterile water for 30 s. Thereafter, each conducted immediately prior to inoculations ensured that sample was split longitudinally and tissue samples were rate of conidial germination exceeded 80%. Each seedling excised from vascular discolourations or—if available— was treated on two opposite points (one at the stem base from living sapwood tissue at the transition zone to necrotic approximately 5 cm above ground level and another on the tissue. Samples were placed (4 samples per Petri dish and opposite side of the stem approximately 10 cm above ground stem section) on 9-cm plastic Petri dishes containing 2% level) applying 2 × 0.5 ml conidial suspension or 2 × 0.5 ml MEA medium (as described above). Plates were sealed with sterile water for the controls. Following inoculation, all seed- Parafilm , labelled with a code for the tree species, tree lings were inspected biweekly for the development of dis- number and section code and incubated at 22–24 °C in the ease symptoms until the end of the vegetation period. Dis- dark for 12 days. Resulting isolates morphologically resem- ease severity was rated according to a slightly adapted scale bling V. nonalfalfae (Domsch et al. 1980; Inderbitzin et al. used by Bejarano-Alcázar et al. (1996) and Schall and Davis 2011) were subcultured on the same conditions for another (2009): 0 = no symptoms/healthy leaves, 1 = 1–33% foli- 18 days. Isolated fungal species other than V. nonalfalfae age affected/chlorotic leaves, 2 = 34–66% foliage affected/ were not further examined. A seedling was scored positive necrotic leaf margins, 3 = 67–99% foliage affected/wilting for V. nonalfalfae if the pathogen was reisolated from at least leaves, 4 = dead or defoliated seedling. one of the three sampled sections. 1 3 200 European Journal of Forest Research (2018) 137:197–209 Fig. 1 Discolourations in sap- wood of inoculated seedlings: a A. campestre; b A. pseudoplata- nus; c F. excelsior; d F. pennsyl- vanica; e P. nigra; f Q. robur; g R. pseudoacacia; h T. cordata; i U. laevis; j U. minor Photographs of vascular/sapwood discolourations were listed as susceptible to Verticillium spp. according to Engel- taken from the cross section and longitudinal section hard (1957), Himelick (1969), Pegg and Brady (2002) or (Fig. 1a–j) of representative samples of each species. If pos- Sinclair and Lyon (2005) (Table 1). However, observations sible, photographs were taken from the part opposite of the were not confirmed by a systematic survey on these sites. inoculation site, in order to avoid discolourations caused by embolism, following wound inoculation. In addition to the inoculation experiment on potted seed- Results lings, three sites within Ailanthus-inoculated or natural- infected forest stands were examined in summer 2015 for External disease symptoms and mortality wilting symptoms on non-target tree species: (1 + 2) two on seedlings sites in Lower Austria, where 20 Ailanthus trees had been artificially stem-inoculated in August 2011 and in June 2013, Stem inoculations on Ailanthus resulted in a rapid disease respectively, and (3) a forest plot in Gänserndorf (Lower progression on 100% (20 of 20 plants) of the trees in 2014: Austria) that became naturally infected with V. nonalfalfae The seedlings exhibited minor disease symptoms (mean in summer 2011 (Maschek and Halmschlager 2016a). Obser- disease severity [MDS] of 0.3) already 2 weeks post-inoc- vations focused on indigenous woody species, which were ulations (WPI), progressing rapidly reaching a MDS of 3.2 1 3 European Journal of Forest Research (2018) 137:197–209 201 Table 1 Site descriptions of mixed Ailanthus stands artificially inoculated or naturally infected by Verticillium nonalfalfae, as well as admixed tree species, all of which did not develop any disease symptoms Site Inoculation date Sea level Orientation/gradi- Soil type Stocking Composition of Stem Location, province ent level (in tree species (in number tenths) tenths) (N/ha) 1 30.08.2011 145 m Plain/0° Chernozem 0.9 0.7 Ailanthus 2.500 Niederweiden, (clayey to silty altissima Lower Austria to silty-clayey 0.1 Acer pseudo- suspended allu- platanus vial material). 0.1 Robinia pseu- At 30–100 cm doacacia gravel from river 0.1 other tree Danube species (Acer campestre, Sambucus nigra, Tilia cordata, Betula pendula) 2 04.06.2013 270 m South-east, slight Chernozem/Para- 0.4 0.3 Fagus sylvatica 2.000 Schützen am slope/10° rendzina middle- 0.2 Tilia cordata Gebirge, Burgen- to deep grounded 0.2 Ailanthus land altissima 0.2 Quercus cerris 0.1 Carpinus betulus 3 Natural 245 m Plain/0° Loess, mostly 1 0.4 Ailanthus 3.500 Hohenruppersdorf, infection in middle- to altissima Lower Austria 2010/2011 deep grounded 0.2 Fraxinus nutritious brown excelsior earths 0.2 Acer campestre 0.2 other tree species (Corylus avellana, Prunus serotina, Sorbus tormi- nalis) at the end of the vegetation period in 2014, resulting in 85% plants) developed chlorosis/necrosis 6 WPI and another (17 of 20 plants) dead Ailanthus trees by November 2015, 10% of the inoculated seedlings (2 of 20 plants) exhibited when the reisolations were carried out. 0% (0 of 4 plants) the same symptoms 8 WPI. The controls of F. pennsylvan- of the controls developed disease symptoms or died during ica remained asymptomatic and no seedling (inoculated or the evaluation period. control) died until the end of the evaluation period. Contrary to these results, stem-inoculated A. campestre, R. pseudoacacia inoculated in 2013 exhibited no symp- A. pseudoplatanus, U. laevis, U. minor, P. nigra, Q. robur toms until the end of the vegetation period. Regardless of and T. cordata as well as all controls developed no disease these results, 20% (4 of 20 plants) of the inoculated seed- symptoms throughout the observation period in 2013 or lings showed symptoms of dieback in spring/early sum- 2014. mer in the following year and were dead at the date of the No symptoms were also detected on stem-inoculated new inoculations in 2014 (13.07.2014). The same was true F. excelsior during the vegetation period in 2013, however, for 33% (2 of 6 plants) of the controls. However, dieback in 2014 20% (4 of 20 plants) of the inoculated seedlings also occurred on additionally reared untreated seedlings and 25% (1 of 4 plants) of the controls developed chloro- (i.e. seedlings that were neither inoculated with conidial sis, necrosis and curled leaves 6 WPI resulting in a MDS suspension nor with sterile water) in 2013 that served as of 0.13 for the inoculated seedlings and a MDS rating of reserves. Mortality reached 25% (2 of 8 plants) on these 0.25 for the controls at the end of the vegetation period untreated seedlings. in 2014. Regardless these symptoms, none of these seed- Seedlings of R. pseudoacacia inoculated in 2014 again lings died until the end of the evaluation period. Similar exhibited no symptoms until the end of the vegetation symptoms were developed on F. pennsylvanica, which was period but did not show any mortality in the following inoculated only in 2014: 15% of these seedlings (3 of 20 1 3 202 European Journal of Forest Research (2018) 137:197–209 Table 2 Results of the artificial inoculations of seedlings of non-target tree species carried out in 2013 and 2014 that were processed in Novem- ber 2015 Species Year of Seedlings Disease Mortality Vascular dis- Successful Successful reisola- Status inoculation inoculated/ symptoms (%) colourations reisolations tions from stem sec- controls (n) (%) (%)* total (%)* tions (%)* 2013 2014 2013 2014 2013 2014 2013 2014 Inoc. Control Inoc. Control − 5 cm 0 + 10 cm b b Ailanthus altissima – X – 20/4 – 100 – 85 100 0 66 0 66 0 0 S Acer campestre X X 20/6 20/4 0 0 0 0 30 0 85 0 40 50 65 T Acer pseudoplatanus X X 20/6 20/4 0 0 0 0 5 0 95 0 55 75 50 T Fraxinus excelsior X X 20/6 20/4 0 20 0 0 5 0 20 0 10 10 0 PR Fraxinus pennsylvanica – X – 20/4 – 25 – 0 15 0 0 0 0 0 0 R Populus nigra X X 20/6 20/4 0 0 0 0 5 0 10 0 5 5 0 PR Quercus robur – X – 19/4 – 0 – 0 42 0 63 0 21 42 26 T c c Robinia pseudoacacia X X 20/6 20/4 0 20 0 20 85 0 0 0 0 0 0 R Tilia cordata X X 20/6 20/4 0 0 0 0 60 0 25 0 5 15 10 PR Ulmus laevis X X 20/6 20/4 0 0 0 0 100 0 10 0 5 5 0 PR Ulmus minor X X 20/6 20/4 0 0 0 0 60 0 5 0 0 0 5 PR S = susceptible (wilt/mortality, high rate of reisolation), T = tolerant (no wilt/mortality, high rate of reisolation), PR = possibly resistant (no wilt/mortality, low rate of reisolation), R = resistant (no wilt/mortality, no reisolation) Data of columns with headers indicated by a single asterisk (*) refer to seedling inoculations in 2014 Reisolations were carried out on three different sections of the stem: from the inoculation site (0 cm), 5 cm below the inoculation site (− 5 cm) and 10 cm above the inoculation site (+ 10 cm) Reisolations were carried out only on seedlings that still exhibited at least some living stem/root tissue Mortality attributed to other abiotic/biotic reasons spring/early summer. No symptoms were observed on the controls in 2013 and 2014. Vascular discolourations on seedlings Vascular discolourations in the sapwood varied in colour, appearance and in intensity among the inoculated tree spe- cies (Fig. 1a–j). Furthermore, there was also a high varia- tion in the number of seedlings that exhibited such vascu- lar discolourations due to artificial infections between the tested species. In Ailanthus, vascular discolourations were yellowish/orange. However, these symptoms could only be observed on seedlings (3 of 3 plants) that still exhibited at least some living cortex chlorenchyma (Table  2). In con- trast, dead Ailanthus seedlings (85% or 17 of 20 plants) were lacking these typical yellowish/orange discolourations but exhibited brownish, necrotic cortex chlorenchyma (Fig. 2) and sapwood and were additionally characterized by a foul smell. Orange/brownish discolourations within the sapwood were typically for F. excelsior, F. pennsylvanica and P. nigra (Fig. 1c–e). However, in contrast to Ailanthus, discoloura- tions occurred only on 5% (1 of 20 plants) on F. excelsior and P. nigra and on 15% (3 of 20 plants) of the inoculated seedlings of F. pennsylvanica (Table 2). Brownish discol- ourations of the sapwood (Fig. 1f, h) were found on 42% (8 Fig. 2 Brownish, necrotic cortex chlorenchyma and necrotic sapwood of 19 plants) of the inoculated seedlings of Q. robur and on of dead Ailanthus seedlings (red arrows) observed during examina- 60% (12 of 20 plants) of the examined T. cordata-seedlings tions in November 2015 1 3 European Journal of Forest Research (2018) 137:197–209 203 (Table 2). A. campestre and U. minor exhibited brownish/ sections (14.1% at − 5 cm and 15.6% at + 10 cm from the blackish discolourations that were found on 30% (6 of 20 point of inoculation). On only two tree species, reisolations plants) and on 60% (12 of 20 plants), respectively (Table 2; were more successful on a stem section, which was not Fig. 1a, j). The most distinctive vascular symptoms were the the point of inoculation: on A. campestre (65% or 13 of 20 greenish/blackish discolourations found on A. pseudoplata- plants) and U. minor (5% or 1 of 20 plants), both at + 10 cm. nus, R. pseudoacacia and U. laevis (Fig.  1b, g, i). While Field observations in mature stands subjected to inocula- these symptoms were quite rare on A. pseudoplatanus (5% tions/natural infection of Ailanthus canopy trees on three or 1 of 20 plants), they occurred quite commonly on R. pseu- forest sites revealed that there was no formation of wilt- doacacia (85% or 17 of 20 plants) and were consistently ing symptoms on admixed non-target tree species such found on inoculated seedlings of U. laevis (100% or 20 of 20 as A. campestre, A. pseudoplatanus, F. excelsior, P. alba, plants) (Table 2). In contrast, none of the controls exhibited R. pseudoacacia and U. laevis. However, no isolations were vascular discolourations, which could be related to Verticil- carried out from that symptomless admixed tree species. In lium infections (Table 2). contrast, severe wilting symptoms, dieback and mortality were observed on inoculated/naturally infected Ailanthus Reisolations of V. nonalfalfae from seedlings trees as well as on adjacent Ailanthus trees that got infected via roots grafts (Fig. 4). Successful rate of reisolation differed considerably between the tested tree species. It ranged from 95% in A. pseudopla- tanus to 0% in F. pennsylvanica and R. pseudoacacia. Fur- thermore, results revealed that rate of reisolation did not cor- Discussion relate with the intensity of vascular discolourations. Thus, there were species with heavily discoloured sapwood but low Knowing the potential host range of V. nonalfalfae is of great rate of successful reisolation (R. pseudoacacia, U. laevis), importance for a hazard and exposure risk assessment. It but there were also species of which V. nonalfalfae could be is also an inevitable prerequisite to avoid undesirable non- reisolated quite common although vascular discolourations target-effects on associated tree species using that fungus were rare (A. pseudoplatanus) as well as species that were for the biological control of Ailanthus or other invasive tree characterized by both, a low rate of successful reisolation species. and a low rate of seedlings exhibiting vascular discoloura- However, the descriptions of V. nonalfalfae and the mor- tions (Table 2). phologically indistinguishable V. alfalfae, both resembling Due to the fact that 17 of 20 seedlings of Ailanthus were the distantly related V. albo-atrum in morphology, were pro- already dead at the time of processing, V. nonalfalfae could vided just a few years ago (Inderbitzin et al. 2011). Thus, be reisolated from 66% (2 of 3) of the seedlings that still previous host range studies of V. nonalfalfae are rare and exhibited at least some living stem or root tissue but showed mainly focus on agricultural crops (Inderbitzin and Subbarao an advanced stage of dieback and subsequent colonization 2014), ornamentals (Garibaldi et al. 2016) or woody species by saprobionts. V. nonalfalfae could not be reisolated from native to North America (Schall and Davis 2009; Kasson controls of Ailanthus. et al. 2014, 2015). In Europe, V. nonalfalfae was confirmed Besides Ailanthus, reisolations were also successful on for the first time just recently from a woody host (Maschek another 8 of 11 tree species (Table  2): Reisolations were and Halmschlager 2016a). Except for A. platanoides, Prunus most successful on A.  pseudoplatanus (95% or 19 of 20 avium and Sambucus racemosa, host range studies on woody plants) although vascular discolourations were found on plants native to Europe are therefore completely lacking so only 5% of these seedlings, followed by A. campestre with far. 85% (17 of 20 plants) and Q.  robur with 63% (12 of 19 Although a long list of diverse hosts—including also plants). V. nonalfalfae could also be successfully reisolated many tree species—is known for V. albo-atrum s.l. (Pegg from 25% (5 of 20 plants) of the seedlings of T.  cordata and Brady 2002; Sinclair and Lyon 2005; Butin 2011), it is and from 20% (4 of 20 plants) of F. excelsior. Low rates of not possible to relate the host range information from previ- successful reisolations were obtained for P. nigra, U. laevis ous literature on V. albo-atrum s.l. to the newly delimitated (10% or 2 of 20 plants) and U. minor (5% or 1 of 20 plants). Verticillium-species V. albo-atrum s.s., V. alfalfae or V. non- V. nonalfalfae could not be reisolated at all from F. penn- alfalfae (Inderbitzin et al. 2011), as long as molecular data or sylvanica and R. pseudoacacia, although each species was detailed morphological descriptions or cultures are lacking. sampled 240 times. In order to expand the information regarding host range of Reisolations were most successful at the point of inocu- V. nonalfalfae, we tested the susceptibility of eight indig- lation (overall rate of reisolation: 20%), and there was only enous non-target tree species, often associated with Tree of a minor difference between the two other sampled stem Heaven, and two invasive exotic tree species. 1 3 204 European Journal of Forest Research (2018) 137:197–209 In our study, V. nonalfalfae induced characteristic wilt- Rudolph 1931; Carter 1938; Weiss 1940; Engelhard 1957; ing symptoms and high mortality only on the target host Himelick 1969; Pegg and Brady 2002), referring to the for- Ailanthus, whereas limited or no impact was detected on mer V. albo-atrum s.l., also A. campestre, A. pseudoplata- non-target tree species, thus indicating a high degree of nus, as well as other Acer species, were considered suscep- host specificity. In addition, consistent vascular discolora- tible. Thus, differences concerning susceptibility/tolerance tions and the successful reisolation of the applied isolate of woody host species between these prior and recent studies indicate the high susceptibility (S) of Ailanthus seedlings to indicate a narrower host range of V. nonalfalfae compared V. nonalfalfae. Similar results had been obtained by Schall to V. albo-atrum s.l. and Davis (2009) and Kasson et al. (2015), testing the host In contrast to Q. robur, which was considered tolerant range for V. nonalfalfae in the eastern USA. Wilt, dieback in our study, V.  nonalfalfae could not be reisolated from and mortality were also observed on artificially inoculated inoculated northern red oak (Quercus rubra) and chestnut canopy trees of Ailanthus on several sites in eastern Aus- oak (Quercus montana) and has therefore been classified tria (Maschek 2011; Maschek and Halmschlager 2017). as resistant by Schall and Davis (2009) and Kasson et al. The same symptoms were also observed on Ailanthus trees (2015). According to field observations in Ailanthus-inoc- adjacent to the inoculated trees, all of which were considered ulated forest stands, resistance was also suggested by the to be connected via root grafts. Intraspecific transmission latter authors for black oak (Quercus velutina). However, by root crafts has already been demonstrated by O’Neal and no inoculation studies had been performed on this tree spe- Davis (2015). Extensive dieback and mortality of Ailanthus cies. In addition, vascular discolourations were observed canopy trees additionally confirm the high virulence of the twice as much in Q. robur (8 of 19 seedlings) in our study, European V. nonalfalfae-isolate G1/5 (Fig. 4) found in seed- compared to Q. rubra (2 of 10 trees) in the study carried ling inoculations and qualify this pathogen as potential bio- out by Kasson et al. (2015). According to previous papers logical control agent for this invasive species (Maschek and dealing with V. albo-atrum s.l., the genus Quercus includes Halmschlager 2016a). both susceptible and resistant species (Himelick 1969; Pegg In contrast to the high susceptibility of Ailanthus, V. non- and Brady 2002; Sinclair and Lyon 2005). V. albo-atrum s.l. alfalfae had no or only limited impact on the tested non- has so far been recorded on the surface of Q. robur acorns target and the two invasive tree species in this study: Based (Urosevic 1987), but is not mentioned in the detailed host on disease symptoms, secondary symptoms like vascular index of V. albo-atrum s.l. published by Engelhard (1957). discolourations and the rate of successful reisolations, tested In F.  excelsior, P.  nigra, T.  cordata, U.  laevis and tree species were assigned as being tolerant, possibly resist- U.  minor, rate of reisolations was rather low and did not ant or resistant. exceed 15% for each stem section (Table 2). Because these Inoculated A. campestre, A. pseudoplatanus and Q. robur species also did not develop disease symptoms (except for exhibited neither primary disease symptoms (chloroses, F. excelsior) and exhibited no mortality, all five species are necroses or wilt) nor mortality; however, V. nonalfalfae was considered possibly resistant (PR). Disease symptoms on frequently reisolated from inoculated seedlings. Recovery the inoculated seedlings of F. excelsior developed simulta- of the pathogen from these asymptomatic seedlings indi- neously to the same extent on the controls and comprised cates a low level of systemic infection that did not result chlorotic and necrotic leaves with typical swellings on the in symptom development. Thus, these three species were rachises. Visual inspections indicated that these swellings considered tolerant (T) to European V. nonalfalfae-isolate could be attributed to the gall-forming midge Dasineura G1/5 (Table 2). Irrespectively of the high rate of reisolations, fraxini; thus, displayed symptoms were not related to V. non- formation of vascular discolourations was only observed to alfalfae infections. a considerably lesser extent on the examined samples. Simi- Similar results were obtained by Schall and Davis (2009) lar results concerning tolerance to V. nonalfalfae had been for V. nonalfalfae-inoculated seedlings and canopy trees of obtained in a host range study by Kasson et al. (2015) for red white ash (Fraxinus americana), which was considered as maple (Acer rubrum) and sugar maple (Acer saccharum), tolerant (= resistant according to our classification), because but also for Korean evodia (Tetradium daniellii) and Tree of reisolation was unsuccessful in all cases. Susceptibility of Paradise (Simarouba glauca). In contrast, inoculated striped F. excelsior to Verticillium wilt (Sinclair and Lyon 2005) maple (Acer pennsylvanicum) reached similar disease levels was usually attributed to V. dahliae infections in previous as Ailanthus in a study carried out in eastern USA by Schall reports (Hiemstra 1995; Heffer and Regan 1996; Schuring and Davis (2009) and was therefore considered susceptible and van der Schaaf 1999). However, Himelick (1969) also to V.  nonalfalfae (isolate PSU140). Susceptibility to this recovered V. albo-atrum s.l. from branch specimens of black, V.  nonalfalfae isolate was also demonstrated for Norway blue, European, green and white ash and was able to reiso- maple (A. platanoides) and Japanese maple (Acer japonica) late the fungus from all inoculated seedlings except black by Kasson et al. (2015). In prior host lists (compiled by ash, which was not included in his pathogenicity test. Green 1 3 European Journal of Forest Research (2018) 137:197–209 205 ash was also included in the host list of V. albo-atrum s.l. compiled by Engelhard (1957). Among poplar species, susceptibility to V. nonalfalfae was previously only tested for White poplar (P. alba) and Bigtooth aspen (Populus grandidentata) (Kasson et  al. 2015). However, the authors did not provide any results for the two species. Assuming that they did not develop symp- toms and were resistant, these results would be similar to those in our study, in which reisolations and vascular discol- ourations (Fig. 1e) were obtained for P. nigra, but occurred very rarely. In contrast to the results related to V. nonalfalfae, the host range of V. albo-atrum s.l. comprised susceptible Populus species such as Populus tremula (Liese 1933; Sin- clair and Lyon 2005). T. cordata exhibited neither wilt symptoms nor mortality (Table 2), which is similar to the results obtained by Kasson et al. (2015) for Tilia americana—this species was therefore considered resistant. In contrast to this study, V. nonalfal- fae was recovered from a quarter of all inoculated seed- lings. Vascular discolourations were found to a lower extent (60%, Fig. 1h) on T. cordata when compared to the results of T. americana (93%, Kasson et al. 2015). According to Fig. 3 Unspecific chloroses and necroses on F.  pennsylvanica in early September 2014 Sinclair and Lyon (2005), the genus Tilia includes both sus- ceptible and resistant species. In prior papers (Wollenweber 1929) and host lists compiled by Engelhard (1957), Himelick according to Heffer and Regan (1996), Hiemstra and Harris (1998) and Sinclair and Lyon (2005), Verticillium wilt of (1969) and Pegg and Brady (2002), T. cordata is frequently listed as susceptible host for V. albo-atrum s.l. along with F. pennsylvanica is generally associated with wilting leaves that remain greenish or is characterized by necroses; thus, other Tilia-species from Europe and Northern America. Both inoculated Ulmus species displayed neither wilt symptoms were not specific for Verticillium wilt. As there were no typical swellings on the leaf rachises, symptoms on symptoms nor mortality (Table  2), but regardless of the absence of these exterior symptoms, vascular discoloura- F. pennsylvanica could also not be attributed to infestation by D. fraxini, as it was the case in F. excelsior. In fact, symp- tions were commonly found in seedlings of U. laevis (100%, Fig. 1i) and U. minor (60%, Fig. 1j). Nevertheless, V. non- toms more resembled to manganese or magnesium deficien- cies (Sinclair and Lyon 2005; Hartmann et al. 2007; Butin alfalfae was reisolated only rarely from those vascular dis- colourations, indicating that the fungus very likely induced et al. 2010), but since nutritional status of seedlings was not further analysed, symptoms might alternatively be attributed these secondary disease symptoms, but could barely sur- vive in both Ulmus species until reisolations in Nov. 2015 to other biotic or abiotic agents. Regardless of these unspe- cific external symptoms, F.  pennsylvanica also developed (i.e. 14 months post-inoculation). Very similar results were obtained by Kasson et al. (2015) for Ulmus americana and less pronounced orange/brownish vascular discolourations (Fig. 1d). However, V. nonalfalfae could not be reisolated Ulmus  pumila: (1) Both species developed neither wilt symptoms nor mortality, (2) both Ulmus species exhibited from any of those discolourations; thus, F. pennsylvanica was considered resistant (R) to V. nonalfalfae. In contrast, vascular discolourations (U. americana 100%, U. pumila 40%) and (3) V. nonalfalfae could not be reisolated from the genus Fraxinus is listed as susceptible to Verticillium spp. (Himelick 1969; Heffer and Regan 1996; Sinclair and any elm-tree. However, U. americana, Ulmus glabra (Syn.: U. montana), U. minor (Syn.: U. campestris), Ulmus parvi- Lyon 2005; Butin et al. 2010) in older publications, taking not into account the recent taxonomic changes (Inderbitzin folia, Ulmus  procera and Ulmus  rubra are mentioned as being susceptible to V. albo-atrum s.l. in the host lists com- et al. 2011). Symptoms on R. pseudoacacia already developed in early piled by Rudolph (1931), Carter (1938), Engelhard (1957), Himelick (1969) or Pegg and Brady (2002). summer 2014 (before the 2014-inoculation was conducted) and occurred not only on Verticillium-inoculated seedlings, Like in F. excelsior, some F. pennsylvanica seedlings also developed chlorotic and necrotic leaves (Fig. 3). However, but also on the controls as well as on seedlings that were 1 3 206 European Journal of Forest Research (2018) 137:197–209 Fig. 4 Canopy trees in an Ailanthus-inoculated mixed forest stand A. pseudoplatanus, F. excelsior (red arrow), P. alba, R. pseudoacacia, in late August 2015, four years after inoculations; Ailanthus trees U. laevis remained asymptomatic showed mortality (blue arrows), whereas admixed A.  campestre, kept in 2013 in reserve. Thus, symptoms cannot be related are in contrast to prior publications and host lists referring to Verticillium infections but were rather related to drought. to V. albo-atrum s.l. (Goidanich 1935; Carter 1938; Engel- Although vascular discolourations were found to the second hard 1957; Himelick 1969; Pegg and Brady 2002; Sinclair most frequency on R. pseudoacacia, V. nonalfalfae could not and Lyon 2005) in which R.  pseudoacacia is considered be reisolated from any of the inoculated seedlings (Table 2). susceptible. Thus, this tree species was also categorized as resistant to Overall, results regarding vascular discolourations and the Austrian V.  nonalfalfae strain. In contrast, artificial the rate of successful reisolations obtained in our seedling inoculations with V. nonalfalfae conducted by Kasson et al. inoculation experiment possibly overestimate the impact of (2015) in 2009 and 2010 induced wilt on 20 and 80% of the V.  nonalfalfae under field conditions, because a success- inoculated plants, but resulted in only 0 and 10% mortality, ful natural infection by this soilborne pathogen might be respectively. Similar to our results, vascular discolourations counteracted or even prevented by root defences (Blanchette were consistently observed on trees inoculated in 2010, and Biggs 1992), which were bypassed by our stem inocula- whereas no discolourations were detected in trees inocu- tion method (Maschek and Halmschlager 2016b). There are lated in 2009 (Kasson et al. 2015). However, sample size also various effects influencing a natural infection or the in 2009 comprised only five black locust trees. Our results establishment of the pathogen within the host: (1) protec- tive effects of mycorrhizal fungi against Verticillium infec- tions (Karagiannidis et al. 2002), (2) synergistic effects of Symptoms only occurred on seedlings that had been moved—with- out the knowledge of the authors—to an unfavourable sun-exposed root-knot nematodes and Verticillium (Santamour 1992) or area of the institute’s garden in order to provide space for another (3) antagonistic effects of xylem-colonizing bacteria (Hall experiment. All other R. pseudoacacia seedlings remained symptom- et al. 1986). free. Seedlings subjected to drought did not recover until the end of the vegetation period 2014 and were classified as loss. 1 3 European Journal of Forest Research (2018) 137:197–209 207 Open Access This article is distributed under the terms of the Crea- Additionally, the applied spore concentration for sure tive Commons Attribution 4.0 International License (http://creat iveco exceeds the amount of conidia found in natural infections mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- (Pegg and Brady 2002). Thus, results indicating suscepti- tion, and reproduction in any medium, provided you give appropriate bility (which was only the case for Ailanthus in our study) credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. in artificial inoculations have to be treated with caution, whereas the absence of wilting symptoms may anyway indicate tolerance or true resistance. 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Arb Biol Reichsanst Land- 24:190–201 Forstw 17:273–299 1 3

Journal

European Journal of Forest ResearchSpringer Journals

Published: Feb 26, 2018

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