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Ash dieback and contributing factors of forest weakening in provenance tests in the Sumy region

Ash dieback and contributing factors of forest weakening in provenance tests in the Sumy region The aim of this study was to evaluate the health condition of Fraxinus excelsior L. in provenance tests, with special focus on ash dieback (ADB), but taking into account also other causes of its decline. The research was carried out in the provenance tests of F. excelsior in the forest-steppe part of the Sumy region. ADB symptoms were revealed in all provenance tests. For 2012–2019 the health condition index, ADB incidence and severity increased for all provenances except the Steppe. Collar rot was present in all ash provenances. Fungi species were isolated from the stem parts of ash at all provenances. Hymenoscyphus fraxineus induced longest necrotic lesions following wound inoculation of stems of 7–10-years-old plants of European ash in the forest while inoculation with both Cytospora sp. and Diplodia sp. resulted in smaller necroses. The conclusion from other regions about the coincidence the damage of European ash by ADB and collar rots as well as the coincidence the damage of European ash by collar rot and tree colonization by Hylesinus crenatus (Fabricius, 1787) is supported. Key words: Fraxinus excelsior; collar rot; health condition; incidence; severity Editor: Bohdan Konôpka is the susceptibility of different provenances to some 1. Introduction pathogens (Metzler et al. 2012; Stener 2013; McKin- Recently, in many European countries, deterioration of ney et al. 2014; Enderle et al. 2015). Such provenance health condition of Fraxinus excelsior L. stands has been tests of Fraxinus excelsior were created in 1930 under registered (Matsiakh & Kramarets 2014; Goberville et al. the leadership of prof. V. M. Andreiev in the Sumy region 2016; Enderle et al. 2017; Meshkova & Borysova 2017), (Davydenko et al. 2013a). which is largely due to the spread of a new invasive patho- In 2012, inspection of these provenance tests was car- gen Hymenoscyphus fraxineus (T. Kowalski) Baral, Que- ried out with assessment of ash dieback (ADB) incidence loz & Hosoya (Queloz et al. 2011; Baral et al. 2014; Gross and severity as well as the health condition for each prov- et al. 2014). The presence of this fungus in the sympto- enance (Davydenko et al. 2013a). Over the past years, matic shoots of European ash (Fraxinus excelsior) from the progressive development of ash dieback has been the Sumy region of Ukraine was confirmed by molecular registered in many sample plots in different regions of methods (Davydenko et al. 2013b). The occurrence of Ukraine (Davydenko et al. 2019). Therefore, it was the this pathogen in ash shoots with pronounced symptoms basis for repeated assessment of ash dieback parameters of the disease (necrotic spots on the bark, discoloration) in the provenance tests in the Sumy region. was statistically proven. For trees affected, the gradual The aim of this study was to evaluate the health con- death of crowns, the presence of necrotic spots on the dition parameters of Fraxinus excelsior in provenance bark of shoots, leaves and stems, discoloration of wood tests, with special focus on ash dieback, but taking into and shoots, uneven flowering, and premature fall were account also other causes of its decline. One aspect of our registered (Skovsgaard et al. 2010; Metzler et al. 2012; research on ash dieback was aimed at determining fungi Davydenko & Meshkova 2017). associated with ash trees and the ability of certain fungi Provenance tests with long-term history is a very suitable approach to study many issues, among which to cause necrosis on the shoots of European ash saplings. *Corresponding author. Valentyna Meshkova, e-mail: Valentynameshkova@gmail.com V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 Fungal isolation. The wood samples were taken by 2. Material and methods Haglöf increment borer 10 cm or over in length from The research was carried out in the provenance tests of F. excelsior trees randomly. Five samples per each prov- Fraxinus excelsior in the State Enterprise “Trostyanetske enance (25 samples in total) were packed individu- Forest Economy” in the forest-steppe part of the Sumy ally in Falcon tubes, marked and placed into freezer region (50°29'05" N; 34°57'56" E). (−20 °C). After surface sterilization in flame, wood sam- Within these stands, 5 provenance tests of F. excel- ples were placed in Petri dishes containing ca. 30 ml of 3% sior were tested: Poliskyi, Western Forest-Steppe, malt extract agar medium (MEA) and incubated at room Right-Bank Forest-Steppe, Left-Bank Forest-Steppe temperature (ca. 21 °C) in the dark. Petri dishes were and Steppe. checked daily and outgrowing fungal mycelia were sub- From each provenance, 50 living trees of European cultured to new Petri dishes with MEA. Fungal cultures ash were inspected. Besides diameter at breast height were divided into groups based on their morphology and (DBH) several parameters of ash health condition were for species identification representative cultures from additionally assessed in each provenance test. Social each group were subjected to sequencing of the internal position within the stand was assessed according to transcribed spacer of the fungal ribosomal RNA (ITS Kraft classification (class 1 – predominant trees, class rRNA). Isolation of DNA, amplification and sequencing 2 – dominant trees, class 3 – subdominant trees, class followed methods described by Davydenko & Meshkova 4 – suppressed trees and class 5 – dying trees) (Lech et (2017). The thermal cycling was carried out using an al. 2020). Applied Biosystems GeneAmp PCR System 2700 ther- Category of tree health condition was evaluated on a mal cycler (Foster City, CA, USA): initial denaturation range of visual characteristics (crown density and color, step at 95 °C for 5 min. was followed by 35 amplification the presence and proportion of dead branches in the cycles of denaturation at 95 °C for 30 s, annealing at 55 °C crown, etc.) according to National scale (Davydenko et for 30 s, and extension at 72 °C for 30 s and n fi al extension al. 2019). Each tree was referred to one of four categories step at 72 °C for 7 min. Raw sequence data were analyzed of health condition (1st – healthy; 2nd – weakened; 3rd using the SeqMan Pro version 10.0 software from DNA- – severely weakened; 4th – drying up). Health condition STAR package (DNASTAR, Madison, WI, USA). The index (HCI) for each plot was calculated as mean of the criteria used for identification were: sequence coverage health condition categories of studied trees. > 80%; similarity to taxon level 98–100%, similarity to The incidence of ash dieback, collar rots, and Hyles- genus level 94–97%. Some fungal species were detected inus crenatus (Fabricius, 1787) (Coleoptera: Curculioni- by morphological symptoms (fruit bodies, spores, etc.). dae: Scolytinae) galleries was estimated as a proportion Pathogenicity test. In the inoculation tests carried out of living ash trees with characteristic symptoms within in May 2020, the following fungi, considered as puta- the sample from each provenance. tive pathogens, were used: Alternaria alternata (Fr.) Severity of ash dieback for each ash tree was esti- Keissl., Didymella macrostoma (Mont.) Qian Chen & L. mated by score: 0 points – no symptoms, 1 point – the Cai, Hymenoscyphus fraxineus, Epicoccum nigrum Link, presence of several indirect symptoms (uneven leafing, and Fusarium avenaceum (Fr.) Sacc., Cytospora sp. 15 single drying of shoots, individual necroses on healthy and Diplodia sp. (for the last two fungi, only genera were shoots, drying up to 10% of branches; 2 points – the pres- identified). The pathogenicity of these fungi to the F. ence of these symptoms, drying of more than 10% and excelsior plants was determined in the field experiment. up to 50% of shoots and branches, formation necrosis, The stems of 7–10-year-old plants growing in the for- 3 – drying of more than 50% of the crown, discoloration of est were each superficially wounded by removal of bark wood in the area of necroses, the presence of necroses and (0.5 cm diameter) with a razor blade in 8 cm from the drying of leaves, shoots, branches, 4 – damage to 100% of shoot tip. A disc cut from a fungal culture was placed the crown, the presence of necroses even on green shoots on each wound and then covered by Parafilm strip. In and branches, leaves, stems (Metzler et al. 2012). Sever- the field experiment, five trees per isolate were used (35 ity of ash dieback for each provenance was evaluated as trees per all fungal species), whereas 10 control plants an arithmetic average of all trees scores. were inoculated with sterile MEA. Observations of both By epicormic shoots, the trees were rated as follow: necrosis and tree condition (crown dieback) were made 0 – absence of epicormic shoots; 1 – sporadic epicormic at weekly intervals for 4 months. The data were expressed shoots; 2 – multiple epicormic shoots; 3 – total stem as mean lesion length. Re-isolations of the test fungi from coverage with epicormic shoots (Meshkova & Borysova all the inoculated plants were attempted by plating mate- 2017). Each stem collar was examined for the presence or absence of stem necroses and signs of wood decay rial from lesions (if present) or from points of inocula- fungi. The score of wood decay severity (collar rots) tion. The lesion material was taken from sections ca. 5 considered the part of damaged stem circumference: 0 mm both above and below each inoculation point. Fungi points – absence; 1 point – up to 25% of circumference; growing from the material were re-isolated as described 2 points – 26–50%; 3 points – 51–75%; 4 points – over in the above procedures for isolation. 75% of circumference (Davydenko et al. 2019). 114 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 Normality tests, summary statistics, one-way analy- The health condition index significantly increased in sis of variance (ANOVA), Tukey HSD test with a signifi- 2019 compared with 2012 for all provenances except cance level of p < 0.05 were performed. Shapiro-Wilk the Steppe, for average sample from 2.1 to 2.6. test has proved the normality of analyzed parameters In 2012, ADB incidence was the lowest in the Western (ADB, collar rots and H. crenatus incidence and sever- Forest-Steppe provenance (25%), almost twice as high in ity, health condition index) when comparing the prov- Poliskyi and Right-bank Forest-Steppe provenance and enances between each other in 2012 and 2019, and each the highest in the rest provenances (83.3 and 91.7% in provenance in 2012 and 2019. Microsoft Excel software the Left-bank Forest Steppe and Steppe respectively) and statistical software package PAST: Paleontological (Table 3). The differences between these three groups Statistics Software Package for Education and Data of provenances are significant. Analysis (Hammer et al. 2001) were used. Table 3. Comparison of ADB parameters in Fraxinus excelsior provenance tests in 2012 and 2019. ADB severity, 3. Results ADB incidence, [%]±SE Provenance [points] ±SE 2012 2019 2012 2019 The results of the 2012 assessment showed that the diam- Poliskyi 58.3±6.97 a 90.0±6.71 d 0.7±0.09 b 1.8±0.12 d eter (30.3–32.1 cm), Kraft class (2.3–2.6) and health Western Forest-Steppe 25.0±6.12 b 75.0±4.78 e 0.3±0.09 a 1.3±0.09 c condition index (1.8–2.4) of individual provenances did Right-bank Forest Steppe 58.3±6.97 a 70.0±5.12 ae 0.7±0.09 b 1.2±0.12 c Left-bank Forest Steppe 83.3±5.27 c 100.0±0.00 d 0.8±0.05 b 1.8±0.11 d not have statistically significant differences (Table 1). Steppe 91.7±3.91 c 75.0±4.78 ce 1.4±0.09 c 1.3±0.09 c In 2019 assessment, the diameter (31.1–33.2 cm), Total 63.3±3.05 a 82.0±3.84 b 0.8±0.04 b 1.5±0.10 d Note: *Means followed by different letters in each column are significantly different at the 95% Kraft class (2.2–2.6) and health condition index (2.5– confidence level. 2.8) of individual provenances also did not have statisti- cally significant differences (Table 2). In 2019 compared to 2012, ADB incidence increased Statistically significant differences were registered 1.3 times in total, 3 times in the West Forest-Steppe, between the provenances in terms of branch dieback and 1.5 times in Poliskyi and 1.2 times in the Right-bank the presence of epicormic shoots (Table 2). The branch and Left-bank Forest-Steppe provenances, however, dieback was the lowest in the Steppe provenance (5.5%), the increase was significant only for three provenances which is significantly less than in the other provenances, (Table 3). Signic fi ance of increase the ADB incidence for except for the Western forest-steppe. The highest val- Right-bank Forest- Steppe and of its decrease for Steppe ues of branch dieback were assessed in the Poliskyi and provenance was not proved. Right-bank forest-steppe provenances (15.3%), with the In 2012, ADB severity score was the lowest in the maximum values reaching 50% (Poliskyi). Western Forest steppe (0.3 points) and the highest in the The prevalence of epicormic shoots ranged from 0.8 Steppe provenance (1.4 points) (Table 3). The rest three points in the Steppe and Left-Bank Forest-Steppe prov- provenances with ADB severity score 0.7–0.8 points did enances to 1.7 points in the Right-Bank Forest-Steppe. not differ significantly among themselves, but were sig- However, Tukey’s test showed a significant difference nificantly above the minimum and below the maximum only between Right-Bank Forest-Steppe and two above value of this parameter. mentioned provenances (P < 0.05). In 2019 compared with 2012, the DBH increased in In 2019, ADB severity score was the lowest in the all provenances, and the Kraft class a bit increased only Right-bank Forest steppe (1.2 points) and didn’t differ in the Left-bank Forest Steppe and Steppe provenances. statistically from Western Forest-Steppe and Steppe (1.3 Table 1. General parameters of Fraxinus excelsior health condition in different provenance tests (2012). Provenance DBH, [cm] ±SE Kraft class, [points] ±SE Health condition index, [points] ±SE Poliskyi 30.3±1.21 a 2.5±0.08 a 1.8±0.09 a Western Forest-Steppe 31.1±1.21 a 2.5±0.06 a 1.8±0.08 a Right-bank Forest Steppe 31.3±1.30 a 2.6±0.05 a 2.0±0.10 a Left-bank Forest Steppe 32.1±1.39 a 2.3±0.06 a 2.4±0.09 a Steppe 31.6±1.23 a 2.5±0.07 a 2.4±0.09 a Total sample 31.3±0.84 a 2.5±0.06 a 2.1±0.04 a Note: *Means in each column are not significantly different at the 95% confidence level. Table 2. General parameters of Fraxinus excelsior health condition in different provenance tests (2019). DBH, Kraft class, Health condition index, Branch dieback, Epicormic shoots, Provenance [cm] ±SE [points] ±SE [points] ±SE [%] ±SE [points] ± SE Poliskyi 31.1±1.0 a 2.5±0.1 a 2.5±0.1 a 15.3±2.3 a 0.9±0.2 ab Western Forest-Steppe 31.9±1.6 a 2.5±0.1 a 2.6±0.1 a 11.0±1.8 ab 1.1±0.2 ab Right-bank Forest Steppe 32.2±1.6 a 2.6±0.1 a 2.7±0.1 a 15.3±1.5 a 1.7±0.3 b Left-bank Forest Steppe 33.2±1.3 a 2.2±0.1 a 2.8±0.1 a 14.5±2.3 a 0.8±0.2 a Steppe 32.2±1.6 a 2.4±0.1 a 2.6±0.1 a 5.5±1.4 b 0.8±0.2 a Total sample 32.1±0.63 2.4±0.06 2.6±0.05 12.3±0.91 1.0±0.1 Note: *Means followed by different letters in each column are significantly different at the 95% confidence level. 115 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 points each) provenances. The ADB score in Poliskyi and ence between them was not significant. Severity score of Left-bank Forest Steppe provenances was significantly trees colonization by H. crenatus was 0.1–0.3 points with higher (1.7 points each) comparing to rest provenances also insignificant difference (Table 4). (Table 3). The results obtained allowed pooling the data for all ADB severity score significantly increased over the provenances to test the hypothesis about the possible period 2012–2019 for all provenances except Steppe one. coincidence ash dieback and collar rots, ash dieback and Collar rot incidence was the lowest (20–25%) in the tree colonization by H. crenatus, as well as collar rots and Western Forest-Steppe and Right-bank Forest Steppe tree colonization by H. crenatus (Table 5). provenances, and signic fi antly higher (40–45%) in other The χ -test proved the coincidence the damage of provenances. Collar rot severity score varied from 1.2 to European ash by ash dieback and collar rots as well as 1.8 in different provenances but the difference between the coincidence the damage of European ash by collar them was not signic fi ant. Trees colonization by H. crena- rots and tree colonization by H. crenatus and doesn’t sup- tus was absent in Western Forest-Steppe and Right-bank port the hypothesis about the coincidence the damage Forest Steppe provenances. It varied from 5% (Poliskyi of European ash by ash dieback and tree colonization by provenance) to 30% (Steppe provenance) but the differ- H. crenatus (Table 5). Table 4. Incidence and severity of collar rots and Hylesinus crenatus as the most common damages of European ash in prov- enance tests (2019). Collar rots (CR) Hylesinus crenatus (HC) incidence, severity incidence, severity Provenance [%] ±SE [points] ±SE [%] ±SE [points] ±SE Poliskyi 45±11.1 a 1.8±0.21 a 5±4.9 a 0.1±0.05 a Western Forest-Steppe 20±8.9 b 1.3±0.23 a 0 0 Right-bank Forest Steppe 25±9.7 ab 1.2±0.22 a 0 0 Left-bank Forest Steppe 45±11.1 a 1.8±0.16 a 15±8.0 a 0.2±0.08 a Steppe 40±11.0 a 1.3±0.22 a 30±10.2 a 0.3±0.11 a Total sample 35±4.8 a 1.5±0.10 a 10±3.0 a 0.1±0.03 a Note: *Means followed by different letters in each column are significantly different at the 95% confidence level. Table 5. Proportion of European ash trees with mixed damage and statistical analysis of correlation between mixed forms of damage in European ash trees (2019). Proportion of ash trees with Number of inspected trees with the damage Damage type A Damage type B r χ Conclusion about correlation fact A and B A without B B without A neither A nor B symptoms of mixed damage, [%] ADB CR 35±4.8 35 47 0 18 0.34 11.8 proven ADB HC 10±3.0 10 72 0 18 0.16 2.4 not proven CR HC 8±2.7 8 27 2 67 0.32 10.6 proven Notes: χ = 3.84; N = 100; ADB – ash dieback; CR – collar rot; HC – Hylesinus crenatus 0.05 Table 6. Fungi identified on Fraxinus excelsior stem for different provenances. Frequency of isolation, [%] Western Right-bank Left-bank Fungi Poliskyi Steppe All provenances Forest-Steppe Forest Steppe Forest Steppe Alternaria alternata (Fr.) Keissl. 13.04 5.56 0.00 4.08 7.41 6.35 Alternaria sp. 8.70 0.00 18.18 2.04 7.41 4.76 Armillaria sp. 4.35 0.00 9.09 6.12 3.70 3.97 Aureobasidium pullulans (de Bary & Löwenthal) G. Arnaud 0.00 0.00 0.00 0.00 3.70 1.59 Cladosporium sp. 4.35 0.00 9.09 4.08 11.11 6.35 Cytospora sp. 15 0.00 0.00 9.09 8.16 0.00 3.97 Didymella macrostoma (Mont.) Qian Chen & L. Cai 8.70 16.67 0.00 4.08 3.70 5.56 Diplodia sp. 8.70 0.00 0.00 2.04 7.41 3.97 Epicoccum nigrum Link 8.70 5.56 9.09 6.12 3.70 6.35 Fusarium sp. 8.70 5.56 0.00 6.12 0.00 4.76 Fusarium avenaceum (Fr.) Sacc. 0.00 16.67 0.00 0.00 7.41 4.76 Gliocladium sp. 13.04 0.00 0.00 0.00 14.81 5.56 Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya 0.00 5.56 0.00 8.16 0.00 3.97 Hysterographium fraxini (Pers.) De Not. 0.00 0.00 9.09 0.00 14.81 3.97 Phialophora sp. 0.00 5.56 0.00 4.08 0.00 2.38 Mortierella sp. 0.00 0.00 9.09 8.16 0.00 0.79 Mollisia cinerea (Batsch) P. Karst 0.00 0.00 0.00 6.12 0.00 2.38 Penicillium sp. A 0.00 11.11 0.00 8.16 7.41 7.94 Penicillium sp. B 0.00 0.00 9.09 0.00 3.70 0.79 Pezicula cinnamomea (DC.) Sacc. 4.35 0.00 0.00 4.08 0.00 2.38 Phialocephala sp. 0.00 0.00 0.00 2.04 0.00 0.79 Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. 8.70 11.11 0.00 2.04 3.70 4.76 Pseudocamarosporium brabeji (Marinc., M.J. Wingf. & Crous) Crous 0.00 0.00 9.09 2.04 0.00 1.59 Sordaria sp. 0.00 11.11 0.00 4.08 0.00 3.17 Trichoderma viride Pers. 4.35 0.00 0.00 0.00 0.00 1.59 Unidentified sp. 51 0.00 5.56 0.00 4.08 0.00 3.17 Unidentified sp. 58 4.35 0.00 9.09 4.08 0.00 2.38 Unidentified sp. 74 4.35 0.00 9.09 2.04 2.38 2.38 116 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 The 25 wood samples from the ash stem yielded a total The highest beta diversity levels are found between of 126 fungal isolates (23 from provenance Poliskyi (Pol), Western Forest-Steppe and Right-bank Forest Steppe 18 from provenance Western Forest-Steppe (WF), 11 groups as well as Poliskyi and Right-bank Forest Steppe from provenance Right-bank Forest Steppe (RBFS), 47 groups. NMDS picture groups samples into rough clus- from provenance Left-bank Forest Steppe (LBFS) and 27 ters corresponding to either their geography (Poliskyi, from provenance Steppe (S) respectively), representing Western Forest-Steppe, Right-bank Forest Steppe, 28 taxons, although only 12 of them could be identified etc.). A similar pattern can be seen in the plot (Fig. 2). to species level (Table 6). The Right-bank Forest Steppe sample is a diverse set of Only Epicoccum nigrum was isolated from all five fungal species in comparison to the Poliskyi and Western provenances. From among all fungi, Penicillium sp. A, Forest-Steppe samples that are only a few close associ- Epicoccum nigrum, Cladosporium sp. and A. alternata ated fungi with F. excelsior. The fungi set from Poliskyi occurred most frequently. samples groups more closely with Steppe samples than Four of the fungal species were isolated only from a the Western Forest-Steppe, Right-bank Forest Steppe single provenance: Aureobasidium pullulans (Steppe), and Left-bank Forest Steppe samples (despite the close Mollisia cinerea, Phialocephala sp. (Left-bank Forest geographical proximity of Right-bank Forest Steppe and Steppe), Trichoderma viride (Poliskyi). Each provenance Left-bank Forest Steppe), thus there seems to be strong has a large number of unique species but also a number of influence of provenance on fungal community composi- shared species. The highest diversity in terms of species tion. Left-bank Forest Steppe is the sample with much richness and Shannon’s index was found for the Left- higher species richness than the others. Bank Forest Steppe (Fig. 1). Pathogenicity tests. Inoculation with H. fraxineus, Relatively high species richness was also found for Cytospora sp. and Diplodia sp. isolates was followed the Poliskyi and Steppe provenances. Lowest species by the development of brownish lesions on all stems of richness was found in the Right-bank Forest Steppe. 7–10-year-old plants. The lesions were already obvious However, all groups had relatively higher Shannon index after 4 months and significantly longer than control and values (comparable to samples from Right-bank Forest other isolates. The inoculation trials showed that there Steppe). For species richness values, all pairwise com- were signic fi ant differences in the length of necroses (F = parisons are significantly different (p < 0.05) with Tukey 5.60, p = 0.018) due to the H. fraxineus between the three tests except Western Forest-Steppe and Right-bank For- inoculated species. The mean wood necrosis length on est Steppe; and for Wilcoxon test all are significantly the branches of H. fraxineus was 25.6 ± 1.5 mm (mean different (p < 0.05) except Western Forest-Steppe and ± standard error) while Cytospora sp. and Diplodia sp. Right-bank Forest Steppe. For Shannon index values 11.7 ±1.1 mm and 6.7 ± 1.3 mm, respectively. Moreover, no pairwise comparisons are significantly different with among both the upper and lower lesions, some showed a either the Tukey or Wilcoxon test. superficial splitting of the bark. None of the inoculated Beta diversity statistics/comparisons among the sam- ples were performed with non-metric multi-dimensional plants died within 4 months. In re-isolation, Cytospora scaling (NMDS) analysis (Fig. 2). Figure 2 shows a map sp. and Diplodia sp. were obtained from necrotic sections for beta diversity with unweighted unifrac distances of all inoculated stems of H. fraxineus. among groups. Fig. 1. Box plots of Shannon indices in the five different groups (Pol – Poliskyi, WF – Western Forest Steppe, RBFS – Right-bank Forest Steppe, LBFS – Left-bank Forest Steppe, St – Steppe). 117 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 Fig. 2. NMDS plot of fungal species from Pol – Poliskyi, WF – Western Forest Steppe, RBFS – Right-bank Forest Steppe, LBFS – Left-bank Forest Steppe, St – Steppe. Legends on the plot corresponds to Alt.alt – Alternaria alternate; Alt – Alternaria sp.; Armil – Armillaria sp; Aurebas – Aureobasidium pullulans; Clad – Cladosporium sp; Cytosp – Cytospora sp. 15; Did macr – Didymella macrostoma; Dipl – Diplodia sp.; Epic – Epicoccum nigrum; Fus – Fusarium sp.; Fusaven – Fusarium avenaceum; Gliocl – Gliocladium sp.; Hfrax – Hymenoscyphus fraxineus; Hyster – Hysterographium fraxini; Phial – Phialophora sp; Mort – Mortierella sp.; Molsin – Mollisia cinerea; Pen A – Penicillium sp. A; Pen B – Penicillium sp. B; Pez cin – Pezicula cinnamomea; Phialc – Phialocephala sp.; Sord – Sordaria fimicola; Pseud – Pseudocamarosporium brabeji; Trich – Trichoderma viride; sp 51 – Unidentified sp. 51; sp 58 – Unidentified sp. 58; sp 74 – Unidentified sp. 74. In other cases, the lesion extension was not observed; research attempted to test this hypothesis in the plots of however, the slight yellowing of leaves was noted after 4 provenance tests of Fraxinus excelsior which were created months from inoculation. A. alternata, Didymella mac- in 1930 under the leadership of prof. V.M. Andreiev in Sumy region (Davydenko et al. 2013a). rostoma and Fusarium avenaceum produced no symp- Provenance tests as objects of tree breeding were cre- toms on stems of plants in experiments although all these ated primarily to determine the most adapted and produc- species induced brown discoloration (ca. 1 mm) around tive provenances for specic fi growing conditions in order the inoculation points on 7–10-year-old plants. These to regionalize the supply of seeds for reforestation. The fungi were re-isolated from the points of inoculation. provenances originate from seeds collected from identi- No lesions developed on any of the control plants and fied stands or regions. However, nowadays they are the the wounds healed within 4 months after inoculation. only natural models for predicting the impact of climate change consequences (Matyas 1996) as well as to study the tree resistance to new pathogens (Pliura et al. 2011; 4. Discussion Metzler et al. 2012). Sometimes, the data from clonal Invasive pathogen Hymenoscyphus fraxineus spread in plantations are also considered in the study of resistance Europe over 20 years ago (Baral et al. 2014; Gross et (Enderle et al. 2013, 2015). To study the resistance to al. 2014; Enderle et al. 2015). Its presence in the east ash dieback, a provenance trial was established in 2005 of Ukraine, in particular, in the Sumy region, has been in southwest Germany with plots at four sites and eight confirmed by molecular methods (Davydenko et al. provenances of Fraxinus excelsior (Enderle et al. 2013, 2013b). In contrast to the western regions (Matsiakh & 2015). The intensity of the disease in single trees within Kramarets 2014), the disease in the east develops more the provenances varied constantly between completely slowly and does not often lead to the death of trees (Dav- unaffected trees and their mortality. The study highlights ydenko & Meshkova 2017), which may be due to the fea- a high genetic variation in susceptibility and considerable tures of the climate, in particular, with low precipitation genetic potential for resistance breeding in provenances (Hlásny et al. 2014; Krakovska et al. 2017). According from southwestern Germany. to another hypothesis, the origin of ash plays a role in In a Danish clonal trial comprising 39 F. excelsior the susceptibility of trees to fungal infection (Metzler clones established in 2007 with grafts at two sites, mor- et al. 2012; Stener 2013; McKinney et al. 2014). Our tality was about 40% in 2013. The average percentage 118 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 of crown damage increased from 30% in 2007 to over ferences between other provenances were not signic fi ant 60% in 2013, with substantial variation among clones both by the incidence and severity (Table 4). (McKinney et al. 2014). In France, only 8% healthy trees Statistical analysis supported the conclusion from and 88% of the trees with more than 5% crown decline other regions about the coincidence the damage of were observed. Collar lesions were observed on 33% of European ash by ash dieback and collar rots (Langer the trees and were present in almost all plots (Husson 2017; Davydenko et al. 2019) as well as the coincidence et al. 2011). the damage of European ash by collar rots and tree colo- Genetic studies of F. excelsior clones in Denmark and nization by H. crenatus (Meshkova & Borysova 2017) Sweden did not reveal any differences among populations (Table 5). However, the absence of coincidence of ADB in resistance to ADB (McKinney et al. 2014). However, and tree colonization by H. crenatus may be explained by in Lithuania progeny trials established in 2005 at three relatively low incidence of the trees inhabited by H. cre- different sites showed significant differences in resist - natus and the features of this insect to colonize the lowest ance to ADB among populations (Pliura et al. 2011). This part of stem with thick bark (Okolow 1970) while ADB provides good possibilities for the selection of resistant reveals first in the crowns (Skovsgaard et al. 2010). We populations. The health condition of trees was inu fl enced can suppose that the incidence of other ash bark beetles also by site conditions and infection pressure from the inhabiting upper stem and branches, especially H. fraxini surrounding stands. (Panzer 1779) (Pedrosa-Macedo 1979) and H. toranio We compared the health scores of five provenances (Danthoine, 1788) (Graf 1977), can be more closely in 2012 and 2019 by different parameters of growth and associated with ADB. However, the assessment of these health condition. Both in 2012 and in 2019, statistically insects’ galleries is possible only after tree felling. significant differences in DBH, Kraft class, and health In the present study, the detected fungal communities condition index between provenances were not found were largely composed by the generalist saprotrophic and (Table 1, 2). However, in 2019 compared to 2012, the pathogenic fungal taxa (Table 6). Despite that several health condition index signic fi antly increased for all prov - pathogenic taxa including H. fraxineus, Cytospora sp., enances except the Steppe. According to National scale H. fraxini, etc., the fungal community of all provenances (Davydenko et al. 2019), all provenances in 2012 could showed preference towards saprotrophic species. One to be considered as weakened (1.5 < HCI < 2.5), and in 2019 three pathogenic taxa (including A. alternata, E. nigrum, they all were severely weakened (2.5 < HCI < 3.5). Cytospora sp., H. fraxineus and H. fraxini) were found In 2019, a provenance from Steppe had significantly in all provenances. Among these, A. alternata and E. the lowest branch dieback (average proportion of dry nigrum are known as opportunistic weak pathogens or branches in the crowns) (Table 2). even saprophytes with a worldwide distribution. Cyto- At the same time, ADB incidence was the lowest in phora sp. is known as the causative agent of Cytospora the Western Forest-Steppe provenance in 2012 and this canker. Other pathogenic fungi were H. fraxineus and parameter did not differ from Steppe in 2019 (Table 3). H. fraxini which both are known as a widespread ash ADB severity was the highest in Steppe in 2012 but was dieback-fungi causing massive ash decline. So, the among the lowest values in 2019. pathogenic fungi were likely the primary cause of dis- In 2019 compared to 2012, ADB incidence and sever- ease symptoms. Therefore, we suggest that the fungus ity increased for all provenances except Steppe, however H. fraxineus is a primary pathogen killing healthy ash tis- maximal values (1.8 points) were assessed for Poliskyi sues. However, later the fungus is losing the competition and Left-bank Forest Steppe provenances (Table 3). So with other decomposing and saprotrophic fungi being the data obtained do not allow us to draw a conclusion more successful and taking over their domination in dead about the different susceptibility of individual ash prov- tissues. So, it explains why necrotic ash shoots usually enances to the ADB. It may be connected with rather are colonized by other plant pathogens, endophytes and high age of trees (about 90 years old). saprophytes. In different regions, it was previously found out Our results for the study of fungal community struc- that collar rots (Langer 2017; Davydenko et al. 2019) ture in declining shoots of ash trees in the different geo- and stem colonization with bark beetles (Meshkova & graphic provenances as well as inoculation experiment Borysova 2017) often accompany infestations of ash with revealed 28 fungal taxa associated with ash trees. Among ADB. Therefore, in a survey of provenance tests in 2019, them, at least three fungal species showed the ability to the incidence and severity of collar rots and H. crenatus be pathogens that is the valuable point of this research. was assessed (Table 4). Collar rot presence was revealed Next to Hymenoscyphus fraxineus and Hysterographium in all ash provenances with the lowest values in Western fraxini, Cytospora sp. and Diplodia sp. have been con- Forest-Steppe and Right-bank Forest Steppe. Differ- sidered to be involved in serious decline, in combina- ences between provenances in disease severity according tion with abiotic factors. Our study confirmed previous to visual assessment were not signic fi ant. Trees coloniza - results published. Przybył (2002), Kowalski & Łukomska tion by H. crenatus was absent in Western Forest-Steppe (2005), Lygis et al. (2006) presented data of endophytic and Right-bank Forest Steppe provenances, and the dif- fungi on ash branches with the most characteristic fungi 119 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 as we did like Alternaria alternata, Diplodia mutila, Pho- Davydenko, K., Vasaitis, R., Stenlid, J., Menkis, A., mopsis sp., Cladosporium cladosporioides, Cytospora 2013b: Fungi in foliage and shoots of Fraxinus excel- ambiens and Phomopsis controversa. Fungi belonging to sior in eastern Ukraine: a first report on Hymenoscy- Phomopsis and Cytospora genera are pathogens of weak phus pseudoalbidus. Forest Pathology, 43:462–467. trees and are often present in dying shoots (Przybył 2002; Davydenko K., Meshkova V., 2017: The current situa- Kowalski & Łukomska 2005). tion concerning severity and causes of ash dieback From this point of view, the knowledge of the distri- in Ukraine caused by Hymenoscyphus fraxineus. In: bution of pathogenic fungal species associated with F. Vasaitis R., Enderle R. (eds.): Dieback of European excelsior suffering from ash-dieback phenomenon can Ash (Fraxinus spp.) – Consequences and Guidelines be very useful in forest management and diversity con- for Sustainable Management. The Report on Euro- servation programs. pean Cooperation in Science & Technology (COST) Action FP1103 FRAXBACK. SLU – Swedish Insti- tute of Agricultural Sciences, Uppsala, p. 220–227. Davydenko, K., Borysova, V., Shcherbak, O., Kryshtop, 5. Conclusion Ye., Meshkova, V., 2019: Situation and perspectives Provenance tests of European ash of about 90 years old of ash (Fraxinus spp.) in Ukraine: focus on eastern in Sumy region (Eastern Ukraine) are affected by ash border. Baltic Forestry, 25:193–202. dieback (ADB) caused by pathogen Hymenoscyphus frax- Enderle, R., Peters, F., Nakou, A., Metzler, B., 2013: ineus for at least 10 years. Temporal development of ash dieback symptoms For 2012 and 2019 the health condition index, ADB and spatial distribution of collar rots in a provenance incidence and severity increased for all provenances trial of Fraxinus excelsior. European Journal of Forest except the Steppe. However, the data obtained do not Research, 132:865–876. allow us to draw a conclusion about the different suscep- Enderle, R., Nakou, A., Thomas, K., Metzler, B., 2015: tibility of individual ash provenances to the ADB. Susceptibility of autochthonous German Fraxinus Collar rot was present in all ash provenances with the excelsior clones to Hymenoscyphus pseudoalbidus lowest incidence in provenances from Western Forest- is genetically determined. Annals of Forest Science, Steppe and Right-bank Forest Steppe and the absence of 72:183–193. significant differences between provenances in disease Enderle, R., Fussi, B., Lenz, H. D., Langer, G., Nagel, R., severity. Metzler, B., 2017: Ash dieback in Germany: research Hylesinus crenatus was not revealed in Western For- on disease development, resistance and management est-Steppe and Right-bank Forest Steppe provenances, options. In: Vasaitis, R., Enderle, R. (eds.): Dieback and the differences between other provenances were not of European Ash (Fraxinus spp.) – Consequences and significant both by the incidence and severity. Guidelines for Sustainable Management. The Report Fungi isolated from stem of ash trees with varying on European Cooperation in Science & Technology degrees of decline were identified and the pathogenicity (COST) Action FP1103 FRAXBACK. SLU – Swed- of some of them was determined. Among them, Hymeno- ish Institute of Agricultural Sciences, Uppsala, p. scyphus fraxineus, Cytospora sp. and Diplodia sp. induced 89–105. different necrotic lesions. Goberville, E., Hautekèete, N. C., Kirby, R. R., Piquot, Y., The conclusion from other regions about the coinci- Luczak, C., Beaugrand, G., 2016: Climate change and dence the damage of European ash by ash dieback and the ash dieback crisis. Scientific Reports, 6, 35303. collar rots (Matsiakh & Kramarets 2014; Langer 2017) Graf, P., 1977: A contribution on the biology and con- as well as the coincidence the damage of European ash trol of Hylesinus oleiperda F. (Coleopt., Scolytidae) by collar rots and tree colonization by H. crenatus (Dav- on olive in the Tadla (Morocco). 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L., Hlásny, T., Mátyás, C., Seidl, R., Kulla, L., Merganičová, 2013a: Spread of Hymenoscyphus pseudoalbidus – the K., Trombik, J. et al., 2014: Climate change increases pathogen of ash dieback in the Left-bank Ukraine. the drought risk in central European forests: What Forestry & Forest Melioration, 123:140–145. are the options for adaptation? Lesnícky časopis – Forestry Journal, 60:5–18. 120 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 Husson, C., Scala, B., Caël, O., Frey, P., Feau, N., Ioos, Meshkova, V. L., Borysova, V. L., 2017: Damage causes R., Marçais, B., 2011: Chalara fraxinea is an inva- of European ash in the permanent sampling plots sive pathogen in France. European Journal of Plant in Kharkiv region. Forestry and Forest Melioration, Pathology, 130:311–324. 131:179–186. Kowalski T., Łukomska A., 2005: Badania nad zamiera- Metzler, B., Enderle, R., Karopka, M., Topfner, K., niem jesionu (Fraxinus excelsior L.) w drzewostan- Aldinger, E., 2012: Development of ash dieback in ach Nadleśnictwa Włoszczowa. Acta Agrobotanica, a provenance trial on different sites in southern Ger- 59:429–440. many. Allgemeine Forst- und Jagdzeitumg, 183:168– Krakovska, S., Buksha, I., Shvidenko, A., 2017: Climate 180. change scenarios for an assessment of vulnerability Okolow, C., 1970: Hylesinus crenatus: its morphology, of forests in Ukraine in the 21st century. Aerul si Apa. biology, enemies and economic importance. Folia Componente ale Mediului, p. 387–394. Forestalia Polonica, 16:171–200. Langer, G., 2017: Collar rots in forests of Northwest Ger- Pedrosa-Macedo, J. H., 1979: Zur Bionomie, Ökologie many affected by ash dieback. Baltic Forestry, 23:4–19. und Ethologie des Eschenbastkäfers, Leperisinus var- Lech, P., Żółciak, A., Hildebrand, R., 2020: Occurrence ius F. 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Scandinavian Journal of Forest Research, 28:205–216. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Forestry Journal de Gruyter

Ash dieback and contributing factors of forest weakening in provenance tests in the Sumy region

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Publisher
de Gruyter
Copyright
© 2021 Valentyna Meshkova et al., published by Sciendo
ISSN
0323-1046
eISSN
2454-0358
DOI
10.2478/forj-2021-0001
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Abstract

The aim of this study was to evaluate the health condition of Fraxinus excelsior L. in provenance tests, with special focus on ash dieback (ADB), but taking into account also other causes of its decline. The research was carried out in the provenance tests of F. excelsior in the forest-steppe part of the Sumy region. ADB symptoms were revealed in all provenance tests. For 2012–2019 the health condition index, ADB incidence and severity increased for all provenances except the Steppe. Collar rot was present in all ash provenances. Fungi species were isolated from the stem parts of ash at all provenances. Hymenoscyphus fraxineus induced longest necrotic lesions following wound inoculation of stems of 7–10-years-old plants of European ash in the forest while inoculation with both Cytospora sp. and Diplodia sp. resulted in smaller necroses. The conclusion from other regions about the coincidence the damage of European ash by ADB and collar rots as well as the coincidence the damage of European ash by collar rot and tree colonization by Hylesinus crenatus (Fabricius, 1787) is supported. Key words: Fraxinus excelsior; collar rot; health condition; incidence; severity Editor: Bohdan Konôpka is the susceptibility of different provenances to some 1. Introduction pathogens (Metzler et al. 2012; Stener 2013; McKin- Recently, in many European countries, deterioration of ney et al. 2014; Enderle et al. 2015). Such provenance health condition of Fraxinus excelsior L. stands has been tests of Fraxinus excelsior were created in 1930 under registered (Matsiakh & Kramarets 2014; Goberville et al. the leadership of prof. V. M. Andreiev in the Sumy region 2016; Enderle et al. 2017; Meshkova & Borysova 2017), (Davydenko et al. 2013a). which is largely due to the spread of a new invasive patho- In 2012, inspection of these provenance tests was car- gen Hymenoscyphus fraxineus (T. Kowalski) Baral, Que- ried out with assessment of ash dieback (ADB) incidence loz & Hosoya (Queloz et al. 2011; Baral et al. 2014; Gross and severity as well as the health condition for each prov- et al. 2014). The presence of this fungus in the sympto- enance (Davydenko et al. 2013a). Over the past years, matic shoots of European ash (Fraxinus excelsior) from the progressive development of ash dieback has been the Sumy region of Ukraine was confirmed by molecular registered in many sample plots in different regions of methods (Davydenko et al. 2013b). The occurrence of Ukraine (Davydenko et al. 2019). Therefore, it was the this pathogen in ash shoots with pronounced symptoms basis for repeated assessment of ash dieback parameters of the disease (necrotic spots on the bark, discoloration) in the provenance tests in the Sumy region. was statistically proven. For trees affected, the gradual The aim of this study was to evaluate the health con- death of crowns, the presence of necrotic spots on the dition parameters of Fraxinus excelsior in provenance bark of shoots, leaves and stems, discoloration of wood tests, with special focus on ash dieback, but taking into and shoots, uneven flowering, and premature fall were account also other causes of its decline. One aspect of our registered (Skovsgaard et al. 2010; Metzler et al. 2012; research on ash dieback was aimed at determining fungi Davydenko & Meshkova 2017). associated with ash trees and the ability of certain fungi Provenance tests with long-term history is a very suitable approach to study many issues, among which to cause necrosis on the shoots of European ash saplings. *Corresponding author. Valentyna Meshkova, e-mail: Valentynameshkova@gmail.com V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 Fungal isolation. The wood samples were taken by 2. Material and methods Haglöf increment borer 10 cm or over in length from The research was carried out in the provenance tests of F. excelsior trees randomly. Five samples per each prov- Fraxinus excelsior in the State Enterprise “Trostyanetske enance (25 samples in total) were packed individu- Forest Economy” in the forest-steppe part of the Sumy ally in Falcon tubes, marked and placed into freezer region (50°29'05" N; 34°57'56" E). (−20 °C). After surface sterilization in flame, wood sam- Within these stands, 5 provenance tests of F. excel- ples were placed in Petri dishes containing ca. 30 ml of 3% sior were tested: Poliskyi, Western Forest-Steppe, malt extract agar medium (MEA) and incubated at room Right-Bank Forest-Steppe, Left-Bank Forest-Steppe temperature (ca. 21 °C) in the dark. Petri dishes were and Steppe. checked daily and outgrowing fungal mycelia were sub- From each provenance, 50 living trees of European cultured to new Petri dishes with MEA. Fungal cultures ash were inspected. Besides diameter at breast height were divided into groups based on their morphology and (DBH) several parameters of ash health condition were for species identification representative cultures from additionally assessed in each provenance test. Social each group were subjected to sequencing of the internal position within the stand was assessed according to transcribed spacer of the fungal ribosomal RNA (ITS Kraft classification (class 1 – predominant trees, class rRNA). Isolation of DNA, amplification and sequencing 2 – dominant trees, class 3 – subdominant trees, class followed methods described by Davydenko & Meshkova 4 – suppressed trees and class 5 – dying trees) (Lech et (2017). The thermal cycling was carried out using an al. 2020). Applied Biosystems GeneAmp PCR System 2700 ther- Category of tree health condition was evaluated on a mal cycler (Foster City, CA, USA): initial denaturation range of visual characteristics (crown density and color, step at 95 °C for 5 min. was followed by 35 amplification the presence and proportion of dead branches in the cycles of denaturation at 95 °C for 30 s, annealing at 55 °C crown, etc.) according to National scale (Davydenko et for 30 s, and extension at 72 °C for 30 s and n fi al extension al. 2019). Each tree was referred to one of four categories step at 72 °C for 7 min. Raw sequence data were analyzed of health condition (1st – healthy; 2nd – weakened; 3rd using the SeqMan Pro version 10.0 software from DNA- – severely weakened; 4th – drying up). Health condition STAR package (DNASTAR, Madison, WI, USA). The index (HCI) for each plot was calculated as mean of the criteria used for identification were: sequence coverage health condition categories of studied trees. > 80%; similarity to taxon level 98–100%, similarity to The incidence of ash dieback, collar rots, and Hyles- genus level 94–97%. Some fungal species were detected inus crenatus (Fabricius, 1787) (Coleoptera: Curculioni- by morphological symptoms (fruit bodies, spores, etc.). dae: Scolytinae) galleries was estimated as a proportion Pathogenicity test. In the inoculation tests carried out of living ash trees with characteristic symptoms within in May 2020, the following fungi, considered as puta- the sample from each provenance. tive pathogens, were used: Alternaria alternata (Fr.) Severity of ash dieback for each ash tree was esti- Keissl., Didymella macrostoma (Mont.) Qian Chen & L. mated by score: 0 points – no symptoms, 1 point – the Cai, Hymenoscyphus fraxineus, Epicoccum nigrum Link, presence of several indirect symptoms (uneven leafing, and Fusarium avenaceum (Fr.) Sacc., Cytospora sp. 15 single drying of shoots, individual necroses on healthy and Diplodia sp. (for the last two fungi, only genera were shoots, drying up to 10% of branches; 2 points – the pres- identified). The pathogenicity of these fungi to the F. ence of these symptoms, drying of more than 10% and excelsior plants was determined in the field experiment. up to 50% of shoots and branches, formation necrosis, The stems of 7–10-year-old plants growing in the for- 3 – drying of more than 50% of the crown, discoloration of est were each superficially wounded by removal of bark wood in the area of necroses, the presence of necroses and (0.5 cm diameter) with a razor blade in 8 cm from the drying of leaves, shoots, branches, 4 – damage to 100% of shoot tip. A disc cut from a fungal culture was placed the crown, the presence of necroses even on green shoots on each wound and then covered by Parafilm strip. In and branches, leaves, stems (Metzler et al. 2012). Sever- the field experiment, five trees per isolate were used (35 ity of ash dieback for each provenance was evaluated as trees per all fungal species), whereas 10 control plants an arithmetic average of all trees scores. were inoculated with sterile MEA. Observations of both By epicormic shoots, the trees were rated as follow: necrosis and tree condition (crown dieback) were made 0 – absence of epicormic shoots; 1 – sporadic epicormic at weekly intervals for 4 months. The data were expressed shoots; 2 – multiple epicormic shoots; 3 – total stem as mean lesion length. Re-isolations of the test fungi from coverage with epicormic shoots (Meshkova & Borysova all the inoculated plants were attempted by plating mate- 2017). Each stem collar was examined for the presence or absence of stem necroses and signs of wood decay rial from lesions (if present) or from points of inocula- fungi. The score of wood decay severity (collar rots) tion. The lesion material was taken from sections ca. 5 considered the part of damaged stem circumference: 0 mm both above and below each inoculation point. Fungi points – absence; 1 point – up to 25% of circumference; growing from the material were re-isolated as described 2 points – 26–50%; 3 points – 51–75%; 4 points – over in the above procedures for isolation. 75% of circumference (Davydenko et al. 2019). 114 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 Normality tests, summary statistics, one-way analy- The health condition index significantly increased in sis of variance (ANOVA), Tukey HSD test with a signifi- 2019 compared with 2012 for all provenances except cance level of p < 0.05 were performed. Shapiro-Wilk the Steppe, for average sample from 2.1 to 2.6. test has proved the normality of analyzed parameters In 2012, ADB incidence was the lowest in the Western (ADB, collar rots and H. crenatus incidence and sever- Forest-Steppe provenance (25%), almost twice as high in ity, health condition index) when comparing the prov- Poliskyi and Right-bank Forest-Steppe provenance and enances between each other in 2012 and 2019, and each the highest in the rest provenances (83.3 and 91.7% in provenance in 2012 and 2019. Microsoft Excel software the Left-bank Forest Steppe and Steppe respectively) and statistical software package PAST: Paleontological (Table 3). The differences between these three groups Statistics Software Package for Education and Data of provenances are significant. Analysis (Hammer et al. 2001) were used. Table 3. Comparison of ADB parameters in Fraxinus excelsior provenance tests in 2012 and 2019. ADB severity, 3. Results ADB incidence, [%]±SE Provenance [points] ±SE 2012 2019 2012 2019 The results of the 2012 assessment showed that the diam- Poliskyi 58.3±6.97 a 90.0±6.71 d 0.7±0.09 b 1.8±0.12 d eter (30.3–32.1 cm), Kraft class (2.3–2.6) and health Western Forest-Steppe 25.0±6.12 b 75.0±4.78 e 0.3±0.09 a 1.3±0.09 c condition index (1.8–2.4) of individual provenances did Right-bank Forest Steppe 58.3±6.97 a 70.0±5.12 ae 0.7±0.09 b 1.2±0.12 c Left-bank Forest Steppe 83.3±5.27 c 100.0±0.00 d 0.8±0.05 b 1.8±0.11 d not have statistically significant differences (Table 1). Steppe 91.7±3.91 c 75.0±4.78 ce 1.4±0.09 c 1.3±0.09 c In 2019 assessment, the diameter (31.1–33.2 cm), Total 63.3±3.05 a 82.0±3.84 b 0.8±0.04 b 1.5±0.10 d Note: *Means followed by different letters in each column are significantly different at the 95% Kraft class (2.2–2.6) and health condition index (2.5– confidence level. 2.8) of individual provenances also did not have statisti- cally significant differences (Table 2). In 2019 compared to 2012, ADB incidence increased Statistically significant differences were registered 1.3 times in total, 3 times in the West Forest-Steppe, between the provenances in terms of branch dieback and 1.5 times in Poliskyi and 1.2 times in the Right-bank the presence of epicormic shoots (Table 2). The branch and Left-bank Forest-Steppe provenances, however, dieback was the lowest in the Steppe provenance (5.5%), the increase was significant only for three provenances which is significantly less than in the other provenances, (Table 3). Signic fi ance of increase the ADB incidence for except for the Western forest-steppe. The highest val- Right-bank Forest- Steppe and of its decrease for Steppe ues of branch dieback were assessed in the Poliskyi and provenance was not proved. Right-bank forest-steppe provenances (15.3%), with the In 2012, ADB severity score was the lowest in the maximum values reaching 50% (Poliskyi). Western Forest steppe (0.3 points) and the highest in the The prevalence of epicormic shoots ranged from 0.8 Steppe provenance (1.4 points) (Table 3). The rest three points in the Steppe and Left-Bank Forest-Steppe prov- provenances with ADB severity score 0.7–0.8 points did enances to 1.7 points in the Right-Bank Forest-Steppe. not differ significantly among themselves, but were sig- However, Tukey’s test showed a significant difference nificantly above the minimum and below the maximum only between Right-Bank Forest-Steppe and two above value of this parameter. mentioned provenances (P < 0.05). In 2019 compared with 2012, the DBH increased in In 2019, ADB severity score was the lowest in the all provenances, and the Kraft class a bit increased only Right-bank Forest steppe (1.2 points) and didn’t differ in the Left-bank Forest Steppe and Steppe provenances. statistically from Western Forest-Steppe and Steppe (1.3 Table 1. General parameters of Fraxinus excelsior health condition in different provenance tests (2012). Provenance DBH, [cm] ±SE Kraft class, [points] ±SE Health condition index, [points] ±SE Poliskyi 30.3±1.21 a 2.5±0.08 a 1.8±0.09 a Western Forest-Steppe 31.1±1.21 a 2.5±0.06 a 1.8±0.08 a Right-bank Forest Steppe 31.3±1.30 a 2.6±0.05 a 2.0±0.10 a Left-bank Forest Steppe 32.1±1.39 a 2.3±0.06 a 2.4±0.09 a Steppe 31.6±1.23 a 2.5±0.07 a 2.4±0.09 a Total sample 31.3±0.84 a 2.5±0.06 a 2.1±0.04 a Note: *Means in each column are not significantly different at the 95% confidence level. Table 2. General parameters of Fraxinus excelsior health condition in different provenance tests (2019). DBH, Kraft class, Health condition index, Branch dieback, Epicormic shoots, Provenance [cm] ±SE [points] ±SE [points] ±SE [%] ±SE [points] ± SE Poliskyi 31.1±1.0 a 2.5±0.1 a 2.5±0.1 a 15.3±2.3 a 0.9±0.2 ab Western Forest-Steppe 31.9±1.6 a 2.5±0.1 a 2.6±0.1 a 11.0±1.8 ab 1.1±0.2 ab Right-bank Forest Steppe 32.2±1.6 a 2.6±0.1 a 2.7±0.1 a 15.3±1.5 a 1.7±0.3 b Left-bank Forest Steppe 33.2±1.3 a 2.2±0.1 a 2.8±0.1 a 14.5±2.3 a 0.8±0.2 a Steppe 32.2±1.6 a 2.4±0.1 a 2.6±0.1 a 5.5±1.4 b 0.8±0.2 a Total sample 32.1±0.63 2.4±0.06 2.6±0.05 12.3±0.91 1.0±0.1 Note: *Means followed by different letters in each column are significantly different at the 95% confidence level. 115 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 points each) provenances. The ADB score in Poliskyi and ence between them was not significant. Severity score of Left-bank Forest Steppe provenances was significantly trees colonization by H. crenatus was 0.1–0.3 points with higher (1.7 points each) comparing to rest provenances also insignificant difference (Table 4). (Table 3). The results obtained allowed pooling the data for all ADB severity score significantly increased over the provenances to test the hypothesis about the possible period 2012–2019 for all provenances except Steppe one. coincidence ash dieback and collar rots, ash dieback and Collar rot incidence was the lowest (20–25%) in the tree colonization by H. crenatus, as well as collar rots and Western Forest-Steppe and Right-bank Forest Steppe tree colonization by H. crenatus (Table 5). provenances, and signic fi antly higher (40–45%) in other The χ -test proved the coincidence the damage of provenances. Collar rot severity score varied from 1.2 to European ash by ash dieback and collar rots as well as 1.8 in different provenances but the difference between the coincidence the damage of European ash by collar them was not signic fi ant. Trees colonization by H. crena- rots and tree colonization by H. crenatus and doesn’t sup- tus was absent in Western Forest-Steppe and Right-bank port the hypothesis about the coincidence the damage Forest Steppe provenances. It varied from 5% (Poliskyi of European ash by ash dieback and tree colonization by provenance) to 30% (Steppe provenance) but the differ- H. crenatus (Table 5). Table 4. Incidence and severity of collar rots and Hylesinus crenatus as the most common damages of European ash in prov- enance tests (2019). Collar rots (CR) Hylesinus crenatus (HC) incidence, severity incidence, severity Provenance [%] ±SE [points] ±SE [%] ±SE [points] ±SE Poliskyi 45±11.1 a 1.8±0.21 a 5±4.9 a 0.1±0.05 a Western Forest-Steppe 20±8.9 b 1.3±0.23 a 0 0 Right-bank Forest Steppe 25±9.7 ab 1.2±0.22 a 0 0 Left-bank Forest Steppe 45±11.1 a 1.8±0.16 a 15±8.0 a 0.2±0.08 a Steppe 40±11.0 a 1.3±0.22 a 30±10.2 a 0.3±0.11 a Total sample 35±4.8 a 1.5±0.10 a 10±3.0 a 0.1±0.03 a Note: *Means followed by different letters in each column are significantly different at the 95% confidence level. Table 5. Proportion of European ash trees with mixed damage and statistical analysis of correlation between mixed forms of damage in European ash trees (2019). Proportion of ash trees with Number of inspected trees with the damage Damage type A Damage type B r χ Conclusion about correlation fact A and B A without B B without A neither A nor B symptoms of mixed damage, [%] ADB CR 35±4.8 35 47 0 18 0.34 11.8 proven ADB HC 10±3.0 10 72 0 18 0.16 2.4 not proven CR HC 8±2.7 8 27 2 67 0.32 10.6 proven Notes: χ = 3.84; N = 100; ADB – ash dieback; CR – collar rot; HC – Hylesinus crenatus 0.05 Table 6. Fungi identified on Fraxinus excelsior stem for different provenances. Frequency of isolation, [%] Western Right-bank Left-bank Fungi Poliskyi Steppe All provenances Forest-Steppe Forest Steppe Forest Steppe Alternaria alternata (Fr.) Keissl. 13.04 5.56 0.00 4.08 7.41 6.35 Alternaria sp. 8.70 0.00 18.18 2.04 7.41 4.76 Armillaria sp. 4.35 0.00 9.09 6.12 3.70 3.97 Aureobasidium pullulans (de Bary & Löwenthal) G. Arnaud 0.00 0.00 0.00 0.00 3.70 1.59 Cladosporium sp. 4.35 0.00 9.09 4.08 11.11 6.35 Cytospora sp. 15 0.00 0.00 9.09 8.16 0.00 3.97 Didymella macrostoma (Mont.) Qian Chen & L. Cai 8.70 16.67 0.00 4.08 3.70 5.56 Diplodia sp. 8.70 0.00 0.00 2.04 7.41 3.97 Epicoccum nigrum Link 8.70 5.56 9.09 6.12 3.70 6.35 Fusarium sp. 8.70 5.56 0.00 6.12 0.00 4.76 Fusarium avenaceum (Fr.) Sacc. 0.00 16.67 0.00 0.00 7.41 4.76 Gliocladium sp. 13.04 0.00 0.00 0.00 14.81 5.56 Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya 0.00 5.56 0.00 8.16 0.00 3.97 Hysterographium fraxini (Pers.) De Not. 0.00 0.00 9.09 0.00 14.81 3.97 Phialophora sp. 0.00 5.56 0.00 4.08 0.00 2.38 Mortierella sp. 0.00 0.00 9.09 8.16 0.00 0.79 Mollisia cinerea (Batsch) P. Karst 0.00 0.00 0.00 6.12 0.00 2.38 Penicillium sp. A 0.00 11.11 0.00 8.16 7.41 7.94 Penicillium sp. B 0.00 0.00 9.09 0.00 3.70 0.79 Pezicula cinnamomea (DC.) Sacc. 4.35 0.00 0.00 4.08 0.00 2.38 Phialocephala sp. 0.00 0.00 0.00 2.04 0.00 0.79 Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. 8.70 11.11 0.00 2.04 3.70 4.76 Pseudocamarosporium brabeji (Marinc., M.J. Wingf. & Crous) Crous 0.00 0.00 9.09 2.04 0.00 1.59 Sordaria sp. 0.00 11.11 0.00 4.08 0.00 3.17 Trichoderma viride Pers. 4.35 0.00 0.00 0.00 0.00 1.59 Unidentified sp. 51 0.00 5.56 0.00 4.08 0.00 3.17 Unidentified sp. 58 4.35 0.00 9.09 4.08 0.00 2.38 Unidentified sp. 74 4.35 0.00 9.09 2.04 2.38 2.38 116 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 The 25 wood samples from the ash stem yielded a total The highest beta diversity levels are found between of 126 fungal isolates (23 from provenance Poliskyi (Pol), Western Forest-Steppe and Right-bank Forest Steppe 18 from provenance Western Forest-Steppe (WF), 11 groups as well as Poliskyi and Right-bank Forest Steppe from provenance Right-bank Forest Steppe (RBFS), 47 groups. NMDS picture groups samples into rough clus- from provenance Left-bank Forest Steppe (LBFS) and 27 ters corresponding to either their geography (Poliskyi, from provenance Steppe (S) respectively), representing Western Forest-Steppe, Right-bank Forest Steppe, 28 taxons, although only 12 of them could be identified etc.). A similar pattern can be seen in the plot (Fig. 2). to species level (Table 6). The Right-bank Forest Steppe sample is a diverse set of Only Epicoccum nigrum was isolated from all five fungal species in comparison to the Poliskyi and Western provenances. From among all fungi, Penicillium sp. A, Forest-Steppe samples that are only a few close associ- Epicoccum nigrum, Cladosporium sp. and A. alternata ated fungi with F. excelsior. The fungi set from Poliskyi occurred most frequently. samples groups more closely with Steppe samples than Four of the fungal species were isolated only from a the Western Forest-Steppe, Right-bank Forest Steppe single provenance: Aureobasidium pullulans (Steppe), and Left-bank Forest Steppe samples (despite the close Mollisia cinerea, Phialocephala sp. (Left-bank Forest geographical proximity of Right-bank Forest Steppe and Steppe), Trichoderma viride (Poliskyi). Each provenance Left-bank Forest Steppe), thus there seems to be strong has a large number of unique species but also a number of influence of provenance on fungal community composi- shared species. The highest diversity in terms of species tion. Left-bank Forest Steppe is the sample with much richness and Shannon’s index was found for the Left- higher species richness than the others. Bank Forest Steppe (Fig. 1). Pathogenicity tests. Inoculation with H. fraxineus, Relatively high species richness was also found for Cytospora sp. and Diplodia sp. isolates was followed the Poliskyi and Steppe provenances. Lowest species by the development of brownish lesions on all stems of richness was found in the Right-bank Forest Steppe. 7–10-year-old plants. The lesions were already obvious However, all groups had relatively higher Shannon index after 4 months and significantly longer than control and values (comparable to samples from Right-bank Forest other isolates. The inoculation trials showed that there Steppe). For species richness values, all pairwise com- were signic fi ant differences in the length of necroses (F = parisons are significantly different (p < 0.05) with Tukey 5.60, p = 0.018) due to the H. fraxineus between the three tests except Western Forest-Steppe and Right-bank For- inoculated species. The mean wood necrosis length on est Steppe; and for Wilcoxon test all are significantly the branches of H. fraxineus was 25.6 ± 1.5 mm (mean different (p < 0.05) except Western Forest-Steppe and ± standard error) while Cytospora sp. and Diplodia sp. Right-bank Forest Steppe. For Shannon index values 11.7 ±1.1 mm and 6.7 ± 1.3 mm, respectively. Moreover, no pairwise comparisons are significantly different with among both the upper and lower lesions, some showed a either the Tukey or Wilcoxon test. superficial splitting of the bark. None of the inoculated Beta diversity statistics/comparisons among the sam- ples were performed with non-metric multi-dimensional plants died within 4 months. In re-isolation, Cytospora scaling (NMDS) analysis (Fig. 2). Figure 2 shows a map sp. and Diplodia sp. were obtained from necrotic sections for beta diversity with unweighted unifrac distances of all inoculated stems of H. fraxineus. among groups. Fig. 1. Box plots of Shannon indices in the five different groups (Pol – Poliskyi, WF – Western Forest Steppe, RBFS – Right-bank Forest Steppe, LBFS – Left-bank Forest Steppe, St – Steppe). 117 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 Fig. 2. NMDS plot of fungal species from Pol – Poliskyi, WF – Western Forest Steppe, RBFS – Right-bank Forest Steppe, LBFS – Left-bank Forest Steppe, St – Steppe. Legends on the plot corresponds to Alt.alt – Alternaria alternate; Alt – Alternaria sp.; Armil – Armillaria sp; Aurebas – Aureobasidium pullulans; Clad – Cladosporium sp; Cytosp – Cytospora sp. 15; Did macr – Didymella macrostoma; Dipl – Diplodia sp.; Epic – Epicoccum nigrum; Fus – Fusarium sp.; Fusaven – Fusarium avenaceum; Gliocl – Gliocladium sp.; Hfrax – Hymenoscyphus fraxineus; Hyster – Hysterographium fraxini; Phial – Phialophora sp; Mort – Mortierella sp.; Molsin – Mollisia cinerea; Pen A – Penicillium sp. A; Pen B – Penicillium sp. B; Pez cin – Pezicula cinnamomea; Phialc – Phialocephala sp.; Sord – Sordaria fimicola; Pseud – Pseudocamarosporium brabeji; Trich – Trichoderma viride; sp 51 – Unidentified sp. 51; sp 58 – Unidentified sp. 58; sp 74 – Unidentified sp. 74. In other cases, the lesion extension was not observed; research attempted to test this hypothesis in the plots of however, the slight yellowing of leaves was noted after 4 provenance tests of Fraxinus excelsior which were created months from inoculation. A. alternata, Didymella mac- in 1930 under the leadership of prof. V.M. Andreiev in Sumy region (Davydenko et al. 2013a). rostoma and Fusarium avenaceum produced no symp- Provenance tests as objects of tree breeding were cre- toms on stems of plants in experiments although all these ated primarily to determine the most adapted and produc- species induced brown discoloration (ca. 1 mm) around tive provenances for specic fi growing conditions in order the inoculation points on 7–10-year-old plants. These to regionalize the supply of seeds for reforestation. The fungi were re-isolated from the points of inoculation. provenances originate from seeds collected from identi- No lesions developed on any of the control plants and fied stands or regions. However, nowadays they are the the wounds healed within 4 months after inoculation. only natural models for predicting the impact of climate change consequences (Matyas 1996) as well as to study the tree resistance to new pathogens (Pliura et al. 2011; 4. Discussion Metzler et al. 2012). Sometimes, the data from clonal Invasive pathogen Hymenoscyphus fraxineus spread in plantations are also considered in the study of resistance Europe over 20 years ago (Baral et al. 2014; Gross et (Enderle et al. 2013, 2015). To study the resistance to al. 2014; Enderle et al. 2015). Its presence in the east ash dieback, a provenance trial was established in 2005 of Ukraine, in particular, in the Sumy region, has been in southwest Germany with plots at four sites and eight confirmed by molecular methods (Davydenko et al. provenances of Fraxinus excelsior (Enderle et al. 2013, 2013b). In contrast to the western regions (Matsiakh & 2015). The intensity of the disease in single trees within Kramarets 2014), the disease in the east develops more the provenances varied constantly between completely slowly and does not often lead to the death of trees (Dav- unaffected trees and their mortality. The study highlights ydenko & Meshkova 2017), which may be due to the fea- a high genetic variation in susceptibility and considerable tures of the climate, in particular, with low precipitation genetic potential for resistance breeding in provenances (Hlásny et al. 2014; Krakovska et al. 2017). According from southwestern Germany. to another hypothesis, the origin of ash plays a role in In a Danish clonal trial comprising 39 F. excelsior the susceptibility of trees to fungal infection (Metzler clones established in 2007 with grafts at two sites, mor- et al. 2012; Stener 2013; McKinney et al. 2014). Our tality was about 40% in 2013. The average percentage 118 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 of crown damage increased from 30% in 2007 to over ferences between other provenances were not signic fi ant 60% in 2013, with substantial variation among clones both by the incidence and severity (Table 4). (McKinney et al. 2014). In France, only 8% healthy trees Statistical analysis supported the conclusion from and 88% of the trees with more than 5% crown decline other regions about the coincidence the damage of were observed. Collar lesions were observed on 33% of European ash by ash dieback and collar rots (Langer the trees and were present in almost all plots (Husson 2017; Davydenko et al. 2019) as well as the coincidence et al. 2011). the damage of European ash by collar rots and tree colo- Genetic studies of F. excelsior clones in Denmark and nization by H. crenatus (Meshkova & Borysova 2017) Sweden did not reveal any differences among populations (Table 5). However, the absence of coincidence of ADB in resistance to ADB (McKinney et al. 2014). However, and tree colonization by H. crenatus may be explained by in Lithuania progeny trials established in 2005 at three relatively low incidence of the trees inhabited by H. cre- different sites showed significant differences in resist - natus and the features of this insect to colonize the lowest ance to ADB among populations (Pliura et al. 2011). This part of stem with thick bark (Okolow 1970) while ADB provides good possibilities for the selection of resistant reveals first in the crowns (Skovsgaard et al. 2010). We populations. The health condition of trees was inu fl enced can suppose that the incidence of other ash bark beetles also by site conditions and infection pressure from the inhabiting upper stem and branches, especially H. fraxini surrounding stands. (Panzer 1779) (Pedrosa-Macedo 1979) and H. toranio We compared the health scores of five provenances (Danthoine, 1788) (Graf 1977), can be more closely in 2012 and 2019 by different parameters of growth and associated with ADB. However, the assessment of these health condition. Both in 2012 and in 2019, statistically insects’ galleries is possible only after tree felling. significant differences in DBH, Kraft class, and health In the present study, the detected fungal communities condition index between provenances were not found were largely composed by the generalist saprotrophic and (Table 1, 2). However, in 2019 compared to 2012, the pathogenic fungal taxa (Table 6). Despite that several health condition index signic fi antly increased for all prov - pathogenic taxa including H. fraxineus, Cytospora sp., enances except the Steppe. According to National scale H. fraxini, etc., the fungal community of all provenances (Davydenko et al. 2019), all provenances in 2012 could showed preference towards saprotrophic species. One to be considered as weakened (1.5 < HCI < 2.5), and in 2019 three pathogenic taxa (including A. alternata, E. nigrum, they all were severely weakened (2.5 < HCI < 3.5). Cytospora sp., H. fraxineus and H. fraxini) were found In 2019, a provenance from Steppe had significantly in all provenances. Among these, A. alternata and E. the lowest branch dieback (average proportion of dry nigrum are known as opportunistic weak pathogens or branches in the crowns) (Table 2). even saprophytes with a worldwide distribution. Cyto- At the same time, ADB incidence was the lowest in phora sp. is known as the causative agent of Cytospora the Western Forest-Steppe provenance in 2012 and this canker. Other pathogenic fungi were H. fraxineus and parameter did not differ from Steppe in 2019 (Table 3). H. fraxini which both are known as a widespread ash ADB severity was the highest in Steppe in 2012 but was dieback-fungi causing massive ash decline. So, the among the lowest values in 2019. pathogenic fungi were likely the primary cause of dis- In 2019 compared to 2012, ADB incidence and sever- ease symptoms. Therefore, we suggest that the fungus ity increased for all provenances except Steppe, however H. fraxineus is a primary pathogen killing healthy ash tis- maximal values (1.8 points) were assessed for Poliskyi sues. However, later the fungus is losing the competition and Left-bank Forest Steppe provenances (Table 3). So with other decomposing and saprotrophic fungi being the data obtained do not allow us to draw a conclusion more successful and taking over their domination in dead about the different susceptibility of individual ash prov- tissues. So, it explains why necrotic ash shoots usually enances to the ADB. It may be connected with rather are colonized by other plant pathogens, endophytes and high age of trees (about 90 years old). saprophytes. In different regions, it was previously found out Our results for the study of fungal community struc- that collar rots (Langer 2017; Davydenko et al. 2019) ture in declining shoots of ash trees in the different geo- and stem colonization with bark beetles (Meshkova & graphic provenances as well as inoculation experiment Borysova 2017) often accompany infestations of ash with revealed 28 fungal taxa associated with ash trees. Among ADB. Therefore, in a survey of provenance tests in 2019, them, at least three fungal species showed the ability to the incidence and severity of collar rots and H. crenatus be pathogens that is the valuable point of this research. was assessed (Table 4). Collar rot presence was revealed Next to Hymenoscyphus fraxineus and Hysterographium in all ash provenances with the lowest values in Western fraxini, Cytospora sp. and Diplodia sp. have been con- Forest-Steppe and Right-bank Forest Steppe. Differ- sidered to be involved in serious decline, in combina- ences between provenances in disease severity according tion with abiotic factors. Our study confirmed previous to visual assessment were not signic fi ant. Trees coloniza - results published. Przybył (2002), Kowalski & Łukomska tion by H. crenatus was absent in Western Forest-Steppe (2005), Lygis et al. (2006) presented data of endophytic and Right-bank Forest Steppe provenances, and the dif- fungi on ash branches with the most characteristic fungi 119 V. Meshkova et al. / Cent. Eur. For. J. 67 (2021) 113–121 as we did like Alternaria alternata, Diplodia mutila, Pho- Davydenko, K., Vasaitis, R., Stenlid, J., Menkis, A., mopsis sp., Cladosporium cladosporioides, Cytospora 2013b: Fungi in foliage and shoots of Fraxinus excel- ambiens and Phomopsis controversa. Fungi belonging to sior in eastern Ukraine: a first report on Hymenoscy- Phomopsis and Cytospora genera are pathogens of weak phus pseudoalbidus. Forest Pathology, 43:462–467. trees and are often present in dying shoots (Przybył 2002; Davydenko K., Meshkova V., 2017: The current situa- Kowalski & Łukomska 2005). tion concerning severity and causes of ash dieback From this point of view, the knowledge of the distri- in Ukraine caused by Hymenoscyphus fraxineus. In: bution of pathogenic fungal species associated with F. Vasaitis R., Enderle R. (eds.): Dieback of European excelsior suffering from ash-dieback phenomenon can Ash (Fraxinus spp.) – Consequences and Guidelines be very useful in forest management and diversity con- for Sustainable Management. The Report on Euro- servation programs. pean Cooperation in Science & Technology (COST) Action FP1103 FRAXBACK. SLU – Swedish Insti- tute of Agricultural Sciences, Uppsala, p. 220–227. Davydenko, K., Borysova, V., Shcherbak, O., Kryshtop, 5. Conclusion Ye., Meshkova, V., 2019: Situation and perspectives Provenance tests of European ash of about 90 years old of ash (Fraxinus spp.) in Ukraine: focus on eastern in Sumy region (Eastern Ukraine) are affected by ash border. Baltic Forestry, 25:193–202. dieback (ADB) caused by pathogen Hymenoscyphus frax- Enderle, R., Peters, F., Nakou, A., Metzler, B., 2013: ineus for at least 10 years. Temporal development of ash dieback symptoms For 2012 and 2019 the health condition index, ADB and spatial distribution of collar rots in a provenance incidence and severity increased for all provenances trial of Fraxinus excelsior. European Journal of Forest except the Steppe. However, the data obtained do not Research, 132:865–876. allow us to draw a conclusion about the different suscep- Enderle, R., Nakou, A., Thomas, K., Metzler, B., 2015: tibility of individual ash provenances to the ADB. Susceptibility of autochthonous German Fraxinus Collar rot was present in all ash provenances with the excelsior clones to Hymenoscyphus pseudoalbidus lowest incidence in provenances from Western Forest- is genetically determined. Annals of Forest Science, Steppe and Right-bank Forest Steppe and the absence of 72:183–193. significant differences between provenances in disease Enderle, R., Fussi, B., Lenz, H. D., Langer, G., Nagel, R., severity. Metzler, B., 2017: Ash dieback in Germany: research Hylesinus crenatus was not revealed in Western For- on disease development, resistance and management est-Steppe and Right-bank Forest Steppe provenances, options. In: Vasaitis, R., Enderle, R. (eds.): Dieback and the differences between other provenances were not of European Ash (Fraxinus spp.) – Consequences and significant both by the incidence and severity. Guidelines for Sustainable Management. The Report Fungi isolated from stem of ash trees with varying on European Cooperation in Science & Technology degrees of decline were identified and the pathogenicity (COST) Action FP1103 FRAXBACK. SLU – Swed- of some of them was determined. Among them, Hymeno- ish Institute of Agricultural Sciences, Uppsala, p. scyphus fraxineus, Cytospora sp. and Diplodia sp. induced 89–105. different necrotic lesions. Goberville, E., Hautekèete, N. C., Kirby, R. R., Piquot, Y., The conclusion from other regions about the coinci- Luczak, C., Beaugrand, G., 2016: Climate change and dence the damage of European ash by ash dieback and the ash dieback crisis. Scientific Reports, 6, 35303. collar rots (Matsiakh & Kramarets 2014; Langer 2017) Graf, P., 1977: A contribution on the biology and con- as well as the coincidence the damage of European ash trol of Hylesinus oleiperda F. (Coleopt., Scolytidae) by collar rots and tree colonization by H. crenatus (Dav- on olive in the Tadla (Morocco). 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Journal

Forestry Journalde Gruyter

Published: Jun 1, 2021

Keywords: Fraxinus excelsior; collar rot; health condition; incidence; severity

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