Journal of Forest Science

JOURNAL OF FOREST SCIENCE

VOLUME 66 ISSUE 6

(On–line) ISSN 1805-935X Prague 2020 (Print) ISSN 1212-4834 Journal of Forest Science continuation of the journal Lesnictví-Forestry

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Volume 66, No. 6 2020

CONTENT

ORIGINAL PAPER

Gallo J., Vacek Z., Baláš M., Vacek S.: Germinative capacity and energy of critically endangered Ojców birch (Betula oycoviensis Besser) in the Czech Republic ...... 227

Ju S., Xu D., Zhang C., Lu J., Jiang X., Ji L.: Induction of tolerance by chlorocholine chloride in Sequoia sempervirens seedlings under natural cooling and drought...... 236

Yousefshahi B., Bazgir M., Jamali S., Kakhki F.V.: Morphological and molecular identification of ectomycorrhizal fungi associated with Persian oak (Quercus brantii Lindl.) tree ...... 244

Yukhnovskyi V., Zibtseva O.: Green space trends in small towns of Kyiv region according to EOS Land Viewer – a case study ...... 252

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Volume 66, No. 6 2020

Editor-in-Chief Vilém V. Podrázský Prague, Czech Republic

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Isabella Børja Libor Jankovský Hans Pretzsch Ås, Norway Brno, Czech Republic Freising, Germany Andrzej Bytnerowicz Vilém Jarský Pasi Puttonen Riverside, California, U.S.A. Prague, Czech Republic Vantaa, Finland Thomas Cech Eric J. Jokela Peter Rademacher Vienna, Austria Gainesville, Florida, U.S.A. Eberswalde, Germany Jan Čermák Dušan Kacálek Jiří Remeš Brno, Czech Republic Opočno, Czech Republic Prague, Czech Republic Jaroslav Červený Jiří Korecký Marcus Schmitt Prague, Czech Republic Prague, Czech Republic Essen, Germany Ignacio J. Diaz-Maroto Emanuel Kula Luděk Šišák Santiago de Compostela, Spain Brno, Czech Republic Prague, Czech Republic Roger-Dirk Eisenhauer Carsten Lorz Vít Šrámek Pirna, Germany Freising, Germany Prague, Czech Republic Michael Englisch Martin Moog Marek Turčáni Vienna, Austria Freising, Germany Prague, Czech Republic Marek Fabrika W. Keith Moser Paweł Tylek Zvolen, Slovak Republic Flagstaff, Arizona, U.S.A. Kraków, Poland Paola Gatto Jindřich Neruda Marcela Van Loo Legnaro, Italy Brno, Czech Republic Vienna, Austria Vladimír Gryc Sasa Orlovic Klaus-Hermann von Wilpert Brno, Czech Republic Novi Sad, Serbia Freiburg, Germany Petr Horáček Aleš Zeidler Brno, Czech Republic Prague, Czech Republic Journal of Forest Science, 66, 2020 (6): 227–235 Original Paper https://doi.org/10.17221/47/2020-JFS

Germinative capacity and energy of critically endangered Ojców birch (Betula oycoviensis Besser) in the Czech Republic

Josef Gallo*, Zdeněk Vacek, Martin Baláš, Stanislav Vacek

Department of Silviculture, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague, Czech Republic *Corresponding author: [email protected]

Citation: Gallo J., Vacek Z., Baláš M., Vacek S. (2020): Germinative capacity and energy of critically endangered Ojców birch (Betula oycoviensis Besser) in the Czech Republic. J. For. Sci., 66: 227–235.

Abstract: Ojców birch (Betula oycoviensis Besser) is a rare and critically endangered taxon of the genus Betula. Its distribution is limited to few countries in Europe. In the Czech Republic, this taxon, characterized by typical shrubby habitus, has been found in fewer than 70 tree individuals, prevailingly in the studied locality Volyně, West Bohemia. This study was focused on the germinative capacity and germinative energy of this taxon, as it is an important indicator of possible regeneration of the trees in nature and for conservation. Non-stratified seeds (2 200 pcs) and stratified ones (2 200 pcs) were compared to each other and in relation to dendrometric tree parameters. The results showed no significant (P > 0.1) differences between stratification variants in total germination (higher by 0.8% in stratified), though stratification improved germinative energy. Germination was in 1st week higher by 6.0% in stratified variant, respectively marginally (P < 0.1) higher in 2nd week in non-stratified variant. However, both germinative capacity and germinative energy were significantly (P < 0.01) variable between individual trees. The germinative capacity was significantly (P < 0.05) positively correlated with tree defoliation. Tiny seeds and triploid trees exhibited very low and zero germinative capacity and energy, respectively. Totally, seeds exhibited sufficient germinative capacity and germinative energy of 23.1% (0.0–62.5%). This suggests that the trees can be potentially used for this type of Ojców birch regeneration i.e. generatively from seeds, although rather in controlled artificial conditions.

Keywords: germination; stratification; silviculture; Ore Mts.; Central Europe

Birches (Betula L.) are an essential ecological the other hand, birch may have commercial impor- component in European forests (Hynynen et al. tance, especially silver birch (Betula pendula Roth) 2010; Vacek et al. 2016). This genus plays an im- and downy birch (Betula pubescens Ehrh.) (Val- portant role in the Czech Republic in terms of in- konen, Valsta 2001; Hynynen et al. 2010). creasing biodiversity, complex forest structure and In Czech Republic, rare taxon of the genus Betula improving soil properties (Podrázský et al. 2009). Ojców birch (Betula oycoviensis Besser) occurs in Birches are light-demanding pioneer tree species, addition to the two species mentioned above (Kar- which rapidly occupy open areas after forest fires, lík et al. 2010; Baláš et al. 2016). Its distribution is post-mining landscape and clear-cuttings due to limited to several countries in Central, Northern their prolific seed production and fast juvenile and Eastern Europe (Staszkiewicz 2013; Vítámvás growth (Fischer et al. 2002; Vacek et al. 2018a). On et al. 2020). At the study area Volyně, Ojców birch

Supported by the Czech University of Life Sciences Prague, Project IGA FLD No. A/19/24 and by the TAČR Agency, Project No. TAČR TH03030339.

227 Original Paper Journal of Forest Science, 66, 2020 (6): 227–235

https://doi.org/10.17221/47/2020-JFS also suffers from high intensity of deer browsing, active management likely to disappear from its im- such as other palatable broadleaved tree species portant locations of occurrence. Seed production (Vacek et al. 2013; Ambrož et al. 2015). Therefore, and subsequent germination play an important the ability (density and growth) of natural regen- role for successful birch reproduction in terms of eration and occurrence of young trees is consid- both natural and artificial regeneration (Hester et erably decreased due to increasing deer popula- al. 1991; Cameron 1996). Moreover, stratification tion in the Czech Republic (Vacek 2017; Cukor et can improve the germinative capacity of this tree al. 2019). Moreover, regeneration of Ojców birch species (Ahola, Leinonen 1999; Mir et al. 2018). For is strictly limited on study site due to dense grass these reasons, presented study is focused on the sward (Baláš et al. 2016). Climatic extremes in re- germination of this rare taxon, as it is an important cent years could also contribute to less vigorous indicator of possible generative regeneration of the growth and regeneration of birch, as it is case of Ojców birch in nature and for conservation. More- many other tree taxons (Leslie et al. 2017; Vacek over, data and research dealing with germination of et al. 2017b; Gallo et al. 2020). On the other hand, Ojców birch are missing. The objectives of this re- birch (especially Carpathian birch [Betula carpati- search were to (1) determine germinative capacity ca W. et K.]) is relatively vulnerable to climatic ex- and germinative energy, (2) compare difference in tremes and air pollution load (Balcar, Kacálek 2001; germination of non-stratified and stratified seeds Novák et al. 2017), that these stresses historically and (3) observe relationship germination, tree pa- caused decline and large-scale disturbance of other rameters and health status of this rare critically en- tree species in study mountain range systems (Král dangered taxon. et al. 2015; Vacek et al. 2017a; Putalová et al. 2019). For silviculture and forest management, it is par- Material and methods ticularly important to differentiate between Betula pendula Roth group and B. pubescens group and Study area. The seed material of Ojców birch was select them into mixtures according to site specifi- collected in Volyně settlement near Chomutov, West cations to ensure best possible vitality of the stands Bohemia, Czech Republic. The locality is situated in future (Linda et al. 2016). B. pubescens (unlike B. in the Ore Mts. of altitude range 711–745 m a.s.l. pendula) can be successfully in terms of silviculture on nutrient-poor abandoned pastures. Area of in- in mixtures with, for example, spruce, as it does not terest is located close to Natural Monument Local- damage spruce crowns by whipping (Poleno et al. ity Ojców birch near Volyně that was established 2009). In this role, B. oycoviensis belongs to the B. in 1986 of size 1.03 ha due to occurrence of criti- pendula Roth and usually it is not differentiated cally endangered Ojców birch. Soil type is dystric (Kuneš et al. 2019). This critically endangered tax- cambisol and prevailing bedrock is schist and on was first described in 1805 (Besser 1809). Ojców gneiss. Mean annual air temperature reaches 6.5 °C birch has prevailingly a shrubby habitus of height and annual sum of precipitation is 700 mm. The around 4–7 m, but on some locations it reaches study territory belongs to humid continental climate dimensions of medium-size trees with maximum characterized by hot and humid summers and cold height up to 20 m with typical morphology, such to severely cold winters (Dfb) according to Köppen as curved and dense branching, dormant buds and climate classification (Köppen 1936), respectively by epicormic shoots (Staszkiewicz, Wójcicki 1992; detailed region Quitt distribution (Quitt 1971) to Baláš et al. 2016). It is evident that there is no po- a cold climatic region and CH 7 subregion. tential commercial use of Ojców birch in forestry, Data collection. Seeds were collected in Septem- however different growing forms of same species ber after seed year 2018 from morphologically typi- increase the genetic biodiversity of forests (Ivetić cal Ojców birches and from those that carried suffi- et al. 2016). cient number of full seeds. Seeds were taken at tree Successful generative reproduction and follow- height 1–4 m from at least 3 whorls and 2 branches. ing natural or artificial regeneration is one of key Dendrometric parameters and health status (folia- factors for successful forest management and sil- tion) of sampled trees were measured (Table 1). vicultural practise (Karlsson 2001; Vacek et al. Diameter at breast height (DBH) was measured by 2017c), such as in given species. Ojców birch is Blue Mantax caliper (Haglöf, Sweden) to an accu- a rare and declining tree taxon, which is without racy of 1 mm. Tree height, base of live crown and

228 Journal of Forest Science, 66, 2020 (6): 227–235 Original Paper https://doi.org/10.17221/47/2020-JFS

Table 1. Characteristics of trees sampled for seed material in the locality Volyně

GPS Altitude Height DBH Crown width Crown base Foliation Tree ID* SQ Northing Easting (m a.s.l.) (m) (cm) (cm) (cm) (%) VOL-24D 50.44233 13.21838 713 5.0 11.5 43.5 3.0 2.5 80 VOL-16D 50.44249 13.21829 716 4.6 8.0 57.5 2.2 1.9 40 VOL-06D 50.44553 13.20653 718 2.3 3.8 60.5 2.3 1.5 55 VOL-03D 50.44485 13.21222 745 11.8 12.4 95.2 4.1 6.5 50 VOL-15D 50.44263 13.21842 717 11.5 10.8 106.5 4.8 6.0 55 VOL-25D 50.44235 13.21862 711 2.5 4.5 55.6 1.5 1.3 35 VOL-30D 50.44251 13.21845 715 6.4 9.2 69.6 2.5 2.2 40 VOL-32D 50.44285 13.21868 714 4.4 6.0 73.3 1.9 2.2 30 VOL-05D 50.44553 13.20652 718 3.6 4.8 75.0 1.9 1.8 60 VOL-07D 50.44554 13.20653 718 3.5 3.5 100.0 1.4 1.6 40 VOL-38T 50.44128 13.22095 686 4.0 3.5 114.3 2.0 1.0 95

*last letter indicates chromosome number: D – diploid, T – triploid; DBH – diameter at breast height, SQ– slenderness quotient (height to diameter ratio) crown width were measured by Vertex ultrasonic germination were tested in STATISTICA 12 (Stat- hypsometer (Haglöf, Sweden) to an accuracy of Soft) using nonparametric Mann-Whitney U test. 0.1 m. Tree foliation (health status indicator) was Kruskal-Wallis ANOVA test was used to compare estimated to the nearest 5% according to methodol- the differences in germination of individual trees. ogy used in the international project of ICP-Forests Spearman correlation was used to determine the and ICP-Focus (Lorenz 1995). Defoliation was cal- relationship with dendrometric characteristics of culated as difference between 100% and foliation. trees (height, DBH, SQ, crown base, crown width), All trees were tested as diploids, while one tree was health status (foliation) and germination. Statistical triploid individual (VOL-38T) (Baláš et al. 2019). differences among data were recorded as follows: The seeds were stored in cooling box (fridge) significant (P < 0.05, P < 0.01), marginal (P < 0.1) at –2 °C. Two variants were tested: non-stratified and non-significant (P > 0.1). The analysis of the and stratified. Seeds of stratified variant were first principal components (PCA) was performed in Ca- placed in sand and stored in cooling box for one noco 5 software (Šmilauer, Lepš 2014) to evaluate month (cold stratification) before testing. Seeds the relationship between germination, stratificati- were not exposed to light prior the experiment. on, tree individuals and tree parameters. Data were For each tree, 4 transparent plastic boxes (with log-transformed and standardized before analysis. 100 seeds in one box) with double-layered moisten The results of multidimensional PCA analysis were filter paper (ČSN 50 04) were prepared. For each visualized in the form of ordination diagram. tree, altogether 400 seeds (200 seeds per variant) were tested, according to ČSN 48 1211 standard. These prepared variants were cultivated in growing RESULTS chamber (Q-Cell, Poland) with conditions according to standard: temperature 30 °C and light for Germinative capacity and energy 8 hours followed by 16 hours in 20 °C and dark. Results of individual trees and variants (stratified Number of germinated seeds was counted week- × non-stratified) summarized together and com- ly as number of healthy and fully developed seeds. pared are in Table 2. In total, 500 seeds (mean 45 Germinative energy was evaluated as the counting ± 37 SD) of non-stratified variant and 518 seeds after first week of experiment. Next three weekly (mean 47 ± 40 SD) of stratified variant germinated evaluations represented the tests of germinative out of initial 2 200 for each variant. Stratified vari- capacity. ant showing higher germination in comparison to Data analysis. The differences between indivi- non-stratified was marked green, and the contrary dual trees and stratification variants in terms of was marked red in the Table 2. In the first count-

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Table 2. Germination per individual Ojców birch trees differentiated by stratification variants – stratified × non- stratified (stratified variant showing higher germination in comparison to non-stratified is marked green, and the contrary is marked red)

st nd rd th Total initial 1 week 2 week 3 week 4 week Tree ID number non- non- non- non- stratified stratified stratified stratified per variant stratified stratified stratified stratified VOL-24D 200 3 0 0 0 0 0 0 0 VOL-16D 200 11 26 1 0 0 0 0 0 VOL-06D 200 71 57 5 5 0 0 0 1 VOL-03D 200 60 58 3 2 0 0 0 0 VOL-15D 200 52 80 4 6 0 0 0 0 VOL-25D 200 58 66 16 2 0 1 0 0 VOL-30D 200 42 53 8 0 0 0 0 0 VOL-32D 200 109 121 10 9 0 0 1 0 VOL-05D 200 18 8 1 1 0 0 1 0 VOL-07D 200 19 21 7 1 0 0 0 0 VOL-38T 200 0 0 0 0 0 0 0 0 Mean 40 45 5 2 0 0 0 0 Median 42 53 4 1 0 0 0 0 SD 30.9 34.6 4.7 2.9 0.0 0.3 0.4 0.3 Total 443 490 55 26 0 1 2 1

SD – standard error ing, stratified variant performed better in 6 out of (P > 0.1), respectively marginally (P < 0.1) higher in 10 cases. In the second counting, it was better only 2nd week in non-stratified variant. In stratified vari- in 3 cases. Statistically, stratification showed no sig- ant, 1st week 88.6% of seeds germinated from the to- nificant (P > 0.1) differences between stratification tal number of germinated seeds, while it was 94.6% variants in germinative capacity (higher by 0.8% in in non-stratified variant. stratified variant), whereas germination was higher Percentage of germinated healthy seeds for whole in 1st week (germinative energy) in stratified variants period of the experiment per each tree is in Figure 1.

70 non-stratified 60 stratified 50

20 Germinated seeds (%) seeds Germinated 10

Tree ID

Figure 1. Percentage of germinated seeds for the whole experiment period per individual Ojców birch trees

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Figure 2. Ordination diagram showing results of principal components analysis of relationship between germination (total, in 1st week, in 2nd week), tree parameters [Height, DBH – diameter at breast height, SQ – slenderness quotient (height to diameter ratio), Crown base, Crown width], health status (Foliation), stratification variants (stratified, non-stratified), set of chromosomes (diploid, triploid) and individual Ojców birch trees

Regardless of variant, most successful in terms of dination diagram was height to diameter ratio. The germinative capacity was tree ID VOL-32D (62.5%) differences between tree individuals and set of birch followed by VOL-25D (35.8%). Least successful was chromosomes (diploid, triploid) were significant, tree ID VOL-24D (0.8%) because of extremely tiny while germination difference between stratified and seeds. Triploid (ID VOL-38T) did not germinate in non-stratified was very low. both variants. Mean germinative capacity for the Total germination was significantly (P < 0.01) 11 evaluated tree individuals was 23.1% (22.7% and correlated especially with germination in 1st week 23.5% for non-stratified and stratified, respectively). (r = 0.99) and in 2nd week (r = 0.80; Table 3). Tree When only diploids were involved, mean germina- parameters (DBH, height, crown width, crown tive capacity was 25.5% (25.0% for non-stratified and base, HDR) have no significant (P > 0.1) effect on 25.9% for stratified seeds). In four tree individuals, germinative capacity and energy, while these pa- stratification improved overall germinative capac- rameters (except HDR) were significantly positively ity, in 6 trees the number was lower for the stratified (P < 0.01) correlated to each other (r = 0.77–0.93). variant. Generally, there were significant (P < 0.01) On the other site, germination was significant- differences in germination between individual birch ly (P < 0.01) negatively correlated with foliation trees from the study site. (r = 0.72).

Relationship between germination, dendromet- Discussion ric parameters and health status PCA results expressing the relationship between Generative reproduction is one of possible ways germinative capacity and energy, tree parameters to regenerate rare tree species (Vincent 1965; Grime and foliation of individual trees are presented in the 2001). However, seed production is dependent on form of ordination diagram in Figure 2. The first many factors such as mast year, climatic factors and ordination axis explains 44.71%, the first two axes soil conditions (Procházková et al. 2002). In natural 77.40% and the four axes together account for 95.40% condition, success of seed reproduction can be signifi- of data variability. The x-axis represents germination cantly limited due to hares (Lepus) and droughts (Rao difference between stratification variants and the y- et al. 2003; Vacek et al. 2018b). On study site Volyně, axis represents the height to diameter ratio. Tree reproduction is also significantly affected by dense height, DBH and crown parameters were positively grass cover (Baláš et al. 2016), which does not allow correlated to each other. Total germination was pos- the rooting of the seeds in the soil and subsequently itively correlated to germination in 1st and 2nd week, it negatively limits seedlings by strong competition while these parameters were significantly negatively (Vacek et al. 2017c). In controlled conditions, it can correlated with foliation, respectively positively with be done with success that is however conditioned by defoliation. The lowest explanatory variable in or- germinative energy and germinative capacity. In our

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Table 3. Spearman correlation matrix of seed germination (germ.), Ojców birch tree characteristic and health status; significant differences (P < 0.01) are marked by asterisk and green colour

Total 1st week 2nd week Crown Crown Height DBH HDR Foliation germ. germ. germ. base width Total germ. 1.00 1st week germ. 0.99* 1.00 2nd week germ. 0.80* 0.73* 1.00 Height –0.04 0.03 –0.16 1.00 DBH 0.11 0.17 –0.06 0.87* 1.00 HDR –0.07 –0.06 0.03 0.18 –0.24 1.00 Foliation –0.66* –0.66* –0.59* 0.09 0.01 0.27 1.00 Crown base 0.20 0.25 0.07 0.87* 0.93* 0.00 –0.05 1.00 Crown width 0.03 0.06 –0.16 0.77* 0.78* 0.02 0.40 0.72* 1.00

DBH – diameter at breast height, HDR – height to diameter ratio (slenderness quotient) study, stratified variant of Ojców birch seeds showed treatment (Schubert 1993), as for Sorbus torminalis L. higher germinative energy (first counting) and over- (Var et al. 2010) or Taxus baccata L. (Melzack et al. all higher germinative capacity. However, the differ- 1982). In this context, the results of our study are sat- ences were relatively small and did not change the isfying, because they suggest the possibility of practi- germinative dynamics of individual trees. The mean cal use of reproduction from seeds. germinative capacity ranged 23.1% which suggests a The ability to manage species-rich and stable mixed possibility of generative reproduction in controlled forests is dependent on many factors like natural re- environment. generation, deer damage (browsing, fraying and bark Seed germinative capacity is highly variable pa- stripping) and management of disturbances (Simon rameter (Hoffman et al. 2005; Debnárová, Šmelková et al. 2010). In some cases, the ability of natural re- 2008). Juntilla (1970) reported germinative capac- generation of a native and important species that is ity around 75% in dwarf birch (Betula nana L.) for enriching forests is limited. Ojców birch in Volyně seeds exposed to alternating temperatures, but only is a typical example. The only confirmed locality in 10–15% for seeds stratified under a constant tem- Czech Republic suffers from heavy deer browsing perature. High variability was reported also for paper and lack of active silvicultural management. Also, birch (Betula papyrifera Marsh.), as from different extreme climatic events could possibly be the partial provenances it reached 11.1–90.8% (Benowicz et al. reason of recent decline (Gallo et al. 2014; Mikulen- 2001). For Betula pendula, most similar taxon, the ka et al. 2020). Individual Ojców birch trees decline germinative capacity was reported to be between in time and natural regeneration has not occurred 17.5–48.75% (Reyes et al. 1997), which is compara- (Ondráček 2008). Controlled generative reproduc- ble to our findings for Betula oycoviensis. Tylkowski tion in combination with artificial regeneration is (2012) emphasized that the germinability of Betula one of possible ways to conserve the unique birch pendula is affected by storage time and temperature. stands (Košut 1982). Other ways include different For the conditions of our methodology, there should types of vegetative reproduction (Vacek et al. 2010; be no negative impacts as seeds were stored around Vítámvás et al. 2020). Our study showed that dif- one year in –2 °C. This species supposedly does not ferent seed lots of Ojców birch contain viable seeds require light pre-treatment for full germinability, as that can be used for regeneration and conservation some other birches like monarch, Japanese or Erman. of this species, in our case the germinative capacity (Bonner, Karrfalt 2008). Stratification is recommend- was 23%. It is possible that under various other strat- ed, but in our case of B. oycoviensis, it did not have ification treatments the germination could be boost- a significant effect on germination characteristics. ed, but in our case the stratification did not play It is reported from numerous studies that particu- a crucial role for the germinative parameters. Vin- larly rare plant and tree species have low germina- cent (1965) also states regarding birch that stratifica- tive capacity, especially without suitable stratification tion is not necessary, but that seed collection must

232 Journal of Forest Science, 66, 2020 (6): 227–235 Original Paper https://doi.org/10.17221/47/2020-JFS be done in the right time and that for good germina- of natural regeneration of this rare tree species. tion the seeds must not dry out. Sufficient natural regeneration of this species is In terms of tree data, germination of the Ojców a prerequisite for the conservation and successful birch was negatively correlated with tree foliage i.e. development of this valuable population. positively with defoliation. Many statistical analyses of time series often show negative correlations be- REFERENCES tween seed production and radial tree growth (Dit- tmar et al. 2003; Mund et al. 2010), which they refer Ahola V., Leinonen K. (1999): Responses of Betula pendula, to as “switching” resource use from vegetative growth Picea abies, and Pinus sylvestris seeds to red/far-red ratios for reproduction, especially in strong mast years. Sim- as affected by moist chilling and germination temperature. ilarly, there was a decrease in foliage in the seed years Canadian Journal of Forest Research, 29: 1709–1717. of European beech compared to the years without Ambrož R., Vacek S., Vacek Z., Král J., Štefančík I. (2015): fructification (Vacek 1987). This switching of resourc- Current and simulated structure, growth parameters and re- es does not necessarily reflect resource constraints or generation of beech forests with different game management compromise between vegetative and reproductive in the Lány Game Enclosure. Forestry Journal, 61: 78–88. growth or foliage. Rather, it shows that fructification Baláš M., Kuneš I., Gallo J., Rašáková N. (2016): Review on responds to external or internal conditions differ- Betula oycoviensis and foliar morphometry of the species ently than radial growth or foliation. This may mean in Volyně, Czech Republic. Dendrobiology, 76: 117–125. that climatic conditions that increase reproductive Baláš M., Kuneš I., Linda R., Gallo J., Petrásek J. (2019): Výskyt growth are unfavourable for vegetative growth (radial jedinců s fenotypovým projevem břízy ojcovské na lokalitě growth and foliation) (Knops et al. 2007, Hirayama Volyně u Výsluní. [Occurrence of individuals showing the et al. 2008). Also, Innes (1994) and Seidling (2007) phenotypic traits of Oyców birch in the locality Volyně report that both the flowering process and the seed u Výsluní.] Soubor specializovaných map s odborným formation, especially in strong seed years, reduce leaf obsahem. Praha, Ministerstvo zemědělství České republiky. biomass. Bonner, Karrfalt (2008) also mentioned that Balcar V., Kacálek D. (2001): Development of European birch excessive seed production is related to deteriorating and Carpathian birch plantations on the ridge part of the crown vitality. On the other hand, no significant cor- Jizerské hory Mts. In: 50 Years of Forestry Research in relation of germination of the birch trees with other Opočno, VÚLHM VS, Sept 12–13, 2001: 193–200. dendrometric data was found. Benowicz A., Guy R., Carlson M.R., El-Kassaby Y.A. (2001): Genetic variation among paper birch (Betula papyrifera Conclusion Marsh.) populations in germination, frost hardiness, gas exchange and growth. Silvae Genetica, 50: 7–12. Ojców birch has an importance in European na- Besser W.S.J.G. (1809): Primitiae Florae Galiciae Austriacae ture as a rare taxon diversifying European forests Utriusque. Wien, Anton Doll Verlag: 400. and increasing diversity of gene pool in genus Betu- Bonner F.T., Karrfalt R.P. (2008): The Woody Plant Seed la. At the locations of its occurrence, its vitality and Manual. Agricultural Handbook No. 727. Washington, numbers are declining. This study of the germina- DC, US Department of Agriculture, Forest Service: 1223. tive capacity and germinative energy tests showed Cameron A. D. (1996): Managing birch woodlands for the that it can be reproduced generatively with success, production of quality timber. Forestry: An International which is however dependent on seed vitality of in- Journal of Forest Research, 69: 357–371. dividual trees. Supposedly triploid individual was Cukor J., Vacek Z., Linda R., Vacek S., Marada P., Šimůnek unambiguously not able to generatively reproduce. V., Havránek F. (2019): Effects of bark stripping on timber Stratification did not show crucial positive effects production and structure of Norway spruce forests in rela- on the germinative capacity and germinative en- tion to climatic factors. Forests, 10: 320. ergy of Ojców birch, even though improving ger- Debnárová G., Šmelková Ľ. (2008): Sesonal fluctuation in minative energy. On the other hand, the seed year germination of short and long – term Norway spruce (Picea in connection with germination had a significantly abies L. Karst.) seeds. Journal of Forest Science, 54: 389–397. negative effect on birch foliation, as favourable Dittmar C., Zech W., Elling W. (2003): Growth variations of weather conditions in recent years have increased common beech (Fagus sylvatica L.) under different climatic reproductive growth at the expense of vegetative and environmental conditions in Europe – a dendroeco- growth. This is a good signal given the possibility logical study. Forest Ecology and Management, 173: 63–78.

233 Original Paper Journal of Forest Science, 66, 2020 (6): 227–235

https://doi.org/10.17221/47/2020-JFS

Fischer A., Lindner M., Abs C., Lasch P. (2002): Vegetation dy- Korczyk A (1967): Rozmieszczenie geograficznebrzozy ojcows- namics in central European forest ecosystems (near-natural kiej (Betula oycoviensis Bess.) [Geographic distribution of as well as managed) after storm events, Folia Geobotanica, Ojców birch (Betula oycoviensis Bess.)]. Ochrona Przyrody 37: 17–32. 32: 133–170. (in Polish) Gallo J., Baláš M., Linda R., Kuneš I. (2020): The effects of Král J., Vacek S., Vacek Z., Putalová T., Bulušek D., Štefančík I. planting stock size and weeding on survival and growth of (2015): Structure, development and health status of spruce small-leaved lime under drought-heat stress in the Czech forests affected by air pollution in the western Krkonoše Mts. Republic. Austrian Journal of Forest Science, 137: 43–66. in 1979–2014. Forestry Journal, 61: 175–187. Grime J.P. (2001): Plant strategies, vegetation processes and Kuneš I., Linda R., Fér T., Karlík P., Baláš M., Ešnerová J., Vítám- ecosystem properties. Chichester, Wiley & Sons: 417. vás J., Bílý J., Urfus T. (2019): Is Betula carpatica genetically Hester A.J., Gimingham C.H., Miles J. (1991): Succession distinctive? A morphometric, cytometric and molecular study from heather moorland to birch woodland. III. Seed of birches in the Bohemian Massif with a focus on Carpathian availability, germination and early growth. The Journal of birch. PLoS ONE, 14: e0224387. Ecology, 79: 329–344. Leslie A., Mencuccini M., Perks M.P. (2017): A resource capture Hirayama D., Nanami S., Itoh A., Yamakura T. (2008): Indi- efficiency index to compare differences in early growth of four vidual resource allocation to vegetative growth and repro- tree species in northern England. iForest: Biogeosciences and duction in subgenus Cyclobalanopsis (Quercus, Fagaceae) Forestry, 10: 397–405. trees. Ecology Research, 23: 451–458. Linda R., Kuneš I., Baláš M., Gallo J. (2017): Morphological vari- Hoffman J., Chválová K., Palátová E. (2005): Lesné semenárst- ability between diploid and tetraploid taxa of the genus Betula vo na Slovensku. Bratislava, PEREX K+K: 193. (in Slovak) L. in the Czech Republic. Journal Forest Science, 63: 531–537. Hynynen J., Niemistö P., Viherä-Aarnio A., Brunner A., Hein Lorenz M. (1995): International co-operative programme on S., Velling P. (2010): Silviculture of birch (Betula pendula assessment and monitoring of air pollution effects on forests Roth and Betula pubescens Ehrh.) in northern Europe. – ICP forests. Water, Air & Soil Pollution, 85: 1221–1226. Forestry, 83: 103–119. Melzack R. N., Watts D. (1982): Variations in seed weight, ger- Innes J.L. (1994): The occurrence of flowering and fruiting mination, and seedling vigour in the yew (Taxus baccata, L.) on individual trees over 3 years and their effects on sub- in England. Journal of Biogeography, 9: 55–63. sequent crown condition. Trees Structure and Function, Mikulenka P., Prokůpková A., Vacek Z., Vacek S., Bulušek D., 8: 139–150. Simon J., Šimůnek V., Hájek V. (2020). Effect of climate and Ivetić V., Devetaković J., Nonić M., Stanković D., Šijačić- air pollution on radial growth of mixed forests: Abies alba Nikolić M. (2016): Genetic diversity and forest repro- Mill. vs. Picea abies (L.) Karst. Central European Forestry ductive material-from seed source selection to planting. Journal, 66: 23–36. iForest-Biogeosciences and Forestry, 9: 801. Mir N.A., Masoodi T.H., Sofi P.A., Mir S.A., Malik A.R. (2018): Junttila O. (1970): Effects of stratification, gibberellic acid Determination of effect of stratification duration and GA 3 and germination temperature on the germination of Betula on germination and growth of Betula utilis D. Don under nana. Physiologia Plantarum, 23: 425–433. temperate conditions of Kashmir Himalayas. Indian Journal Karlík P. (2010): Taxonomická problematika bříz Betula L. of Plant Physiology, 23: 536–542. v České republice se zvláštním zřetelem na drobné taxony Mund M., Kutsch W.L., Wirth C., Kahl T., Knohl A., Skomarkova z okruhu břízy pýřité Betula pubescens agg. In: Prknová H. V.M., Schulze E.D. (2010): The influence of climate and fructi- (ed.): Bříza – strom roku 2010, Kostelec nad Černými lesy, fication on the inter-annual variability of stem growth and net Sept 23, 2010: 61–65. (abstract in English) primary productivity in an old-growth, mixed beech forest. Karlsson M. (2001). Natural regeneration of broadleaved tree Tree Physiology, 30: 689–704. species in southern Sweden. Acta Universitatis Agricultu- Novák J., Špulák O., Souček J., Slodičák M., Dušek D. (2017): rae Sueciae, 196: 44. Potential of birch in the Czech forests. Beiträge zur Jahresta- Knops J.M.H., Koenig W.D., Carmen W.J. (2007): Negative gung, 2017: 110–115. correlation does not imply a tradeoff between growth Ondráček (2008): Plán péče o přírodní památku – Lokalita břízy and reproduction in California oaks. Proceedings of the ojcovské u Volyně na období 2008–2017. Praha, Agentura National Academy of Sciences of the United States of ochrany přírody a krajiny České republiky: 15. (in Czech) America, 104: 16982–16985. Parisod C., Holderegger R., Brochmann C. (2010): Evolutionary Köppen W. (1936): Das geographische System der Klimate, consequences of autopolyploidy. New Phytologist, 186: 5–17. Handbuch der Klimatologie. Berlin, Gebrüder Borntrae- Podrázský V., Remeš J., Hart V., Moser W. K. (2009): Production ger: 44. and humus form development in forest stands established on

234 Journal of Forest Science, 66, 2020 (6): 227–235 Original Paper https://doi.org/10.17221/47/2020-JFS

agricultural lands – Kostelec nad Černými lesy region. Journal Vacek S., Vacek Z., Bílek L., Simon J., Remeš J., Hůnová I., of Forest Science, 55: 299–305. Mikeska M. (2016). Structure, regeneration and growth of Poleno Z., Vacek S., Podrázský V., Remeš J., Štefančík I., Mikeska Scots pine (Pinus sylvestris L.) stands with respect to changing M., Kobliha J., Kupka I., Malík V., Turčáni M., Dvořák J., Zat- climate and environmental pollution. Silva Fennica, 50: 1564. loukal V., Bílek L., Baláš M., Simon J. (2009): Pěstování lesů Vacek S., Černý T., Vacek Z., Podrázský V., Mikeska M., Králíček III. Praktické postupy pěstování lesů. Kostelec nad Černými I. (2017a): Long-term changes in vegetation and site condi- lesy, Lesnická práce: 952. (in Czech) tions in beech and spruce forests of lower mountain ranges of Procházková Z. Bezděčková L. Martincová J., Palátová E. (2002): Central Europe. Forest Ecology and Management, 398: 75–90. Quality of beechnuts from different crop years. Dendrobiol- Vacek S., Vacek Z., Remeš J., Bílek L., Hůnová I., Bulušek D., ogy, 47: 39–42. Putalová T., Král J., Simon J. (2017b): Sensitivity of unmanaged Putalová T., Vacek Z., Vacek S., Štefančík I., Bulušek D., Král relict pine forest in the Czech Republic to climate change and J. (2019): Tree-ring widths as an indicator of air pollution air pollution. Trees, 31: 1599–1617. stress and climate conditions in different Norway spruce for- Vacek S., Vacek Z., Kalousková I., Cukor J., Bílek L., Moser W. est stands in the Krkonoše Mts. Central European Forestry K., Bulušek D., Podrázský V., Řeháček D. (2018b): Sycamore Journal, 65: 21–33. maple (Acer pseudoplatanus L.) stands on former agricultural Quitt E. (1971): Klimatické oblasti Československa. Brno, Geo- land in the Sudetes – evaluation of ecological value and pro- grafický ústav ČSAV: 5. (in Czech) duction potential. Dendrobiology, 79: 61–76. Rao S.J., Iason G.R., Hulbert I.A.R., Daniels M.J., Racey P.A. Vacek Z., Vacek S., Remeš J., Štefančík I., Bulušek D., Bílek D. (2003): Tree browsing by mountain hares (Lepus timidus) in (2013): The structure and model development of stands in young Scots pine (Pinus sylvestris) and birch (Betula pendula) NNR Trčkov – protected landscape area Orlické hory, Czech woodland. Forest Ecology and Management, 176: 459–471. Republic. Forestry Journal, 59: 248–263. Schubert J. (1993): Lagerung und Vorbehandlung von Saatgut Vacek Z. (2017): Structure and dynamics of spruce-beech-fir for- wichtiger Baum- und Straucharten. Eberswalde-Finow, Lande- ests in Nature Reserves of the Orlické hory Mts. in relation to sanstalt fur Ökologie, Bodenordnung und Forsten/Landesamt ungulate game. Central European Forestry Journal, 63: 23–34. fur Agrarordnung Nordrhein-Westfalen (LÖBF): 183. Vacek Z., Bulušek D., Vacek S., Hejcmanova P., Remeš J., Bílek Seidling W. (2007): Signals of summer drought in crown condi- L., Štefančík I. (2017c): Effect of microrelief and vegetation tion data from the German Level I network. European Journal cover on natural regeneration in European beech forests in of Forest Research, 126: 529–544. Krkonoše national parks (Czech Republic, Poland). Austrian Simon J. (2010): Strategie mamagementu lesních území se Journal of Forest Science, 134: 75–96. zvláštním statutem ochrany. Obecná část I. Kostelec nad Vacek Z., Cukor J., Vacek S., Podrázský V., Linda R., Kovařík Černými lesy, Lesnická práce: 568. (in Czech) J. (2018a): Forest biodiversity and production potential of Šmilauer P., Lepš J. (2014): Multivariate analysis of ecological post-mining landscape: opting for afforestation or leaving data using CANOCO 5. Cambridge, Cambridge University it to spontaneous development? Central European Forestry Press: 361. Journal, 64: 116–126. Staszkiewicz J. (2013): Brzoza ojcowska (Betula oycoviensis Valkonen S., Valsta L. (2001): Productivity and economics of Bess.) na górze Skiełek w Beskidzie Wyspowym. Chrońmy mixed two-storied spruce and birch stands in Southern Fin- Przyrodę Ojczystą, 41: 36–41. Available at http://archive.is/ land simulated with empirical models. Forest Ecology and www.brzozanaskielku.republika.pl (in Polish) Management, 140: 133–149. Staszkiewicz J., Wójcicki J. J. (1992): “Betula × oycoviensis” Var M., Bekci B., Dinçer D. (2010): Effect of stratification treat- Besser in the environs of Kraków (S. Poland). Veröffentli- ments on germination of Sorbus torminalis L. Crantz (wild chungen des Geobotanischen Institutes der Eidgenössische service tree) seeds with different origins. African Journal of Technische Hochschule, Stiftung Rübel, in Zürich 107: 94–97. Biotechnology, 9: 5535–5541. Tylkowski T. (2012): Betula pendula seed storage and sowing Vincent G. (1965): Lesní semenářství. Praha, Státní zemědělské pre-treatment: effect on germination and seedling emergence nakladatelství: 307. (in Czech) in container cultivation. Dendrobiology, 67: 49–58. Vítámvás J., Kuneš I., Viehmannová I., Linda R., Baláš M. Vacek S. (1987): Morfologická proměnlivost buku lesního (2020): Conservation of Betula oycoviensis, an endangered v Krkonoších. Zprávy lesnického výzkumu, 32: 1–6. rare taxon, using vegetative propagation methods. iForest- Vacek S., Nosková I., Bílek L., Vacek Z., Schwarz O. (2010): Biogeosciences and Forestry, 13: 107. Regeneration of forest stands on permanent research plots in the Krkonoše Mts. Journal of Forest Science, 56: 541–554. Received: April 15, 2019 Accepted: June 8, 2020

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Induction of tolerance by chlorocholine chloride in Sequoia sempervirens seedlings under natural cooling and drought

Shuming Ju,1, 2* Delan Xu,1 Cuiying Zhang,1 Jiaqi Lu,1 Xueqing Jiang,1 Lingzhen Ji1

1Xuzhou Institute of Technology, Xuzhou Jiangsu, China 2Jiangsu Laboratory of Pollution Control and Resource Reuse, Xuzhou Jiangsu, China *Corresponding author: [email protected]

Citation: Ju S., Xu D., Zhang C., Lu J., Jiang X., Ji L. (2020): Induction of tolerance by chlorocholine chloride in Sequoia sempervirens seedlings under natural cooling and drought. J. For. Sci., 65: 236–243.

Abstract: Two-years-old Sequoia sempervirens seedlings were foliar sprayed once and twice with chlorocholine chloride (CCC) at 0, 100, 500, 1 000, 1 500 and 2 000 mg·l–1. The purpose was to investigate the effect of CCC on the growth and photosynthetic activity of S. sempervirens seedlings under natural cooling and drought in autumn and winter. The findings showed that the increments of plant height and crown diameter were significantly decreased with the increase of chlorocholine chloride concentration, and the increment of root collar diameter, net photosynthetic rate, actual photochemical quantum yield and photosynthetic electron transport rate showed the trend of increasing first and then decreasing, and reached the maximum at concentrations of 1 000~2 000 mg·l–1. There was not a significant difference between two applications and single application. It suggests that 1 000~2 000 mg·l–1 chlorocholine chloride can protect the photosynthetic activity of S. sempervirens seedlings and alleviate the stress induced by low temperatures and drought in autumn and winter.

Keywords: plant growth regulators; coast redwood; growth indicators; physiological condition; environment stress

Chlorocholine chloride (2-chloroethyltrimethyl- In the practice of agriculture and forestry, a suit- ammonium chloride, CCC), as a plant growth in- able application method of CCC is usually selected hibitor, is widely researched and used in plant pro- based on the criteria of minimum restriction of duction (Wang et al. 2010; Wu et al. 2014; Karimi plant growth, minimum physiological and meta- et al. 2014). Earlier studies have shown that CCC bolic damage and significant improvement of plant application can dwarf plant growth and inhibit resistance (Liu et al. 2013; Zhang et al. 2015a). branch extension, but it increases photosynthetic Coast redwood (Sequoia sempervirens Endl.) be- capacity and yield of plant (Wang et al. 2009; Xu longs to relic plants, Taxodiaceae (Zuo et al. 2000; et al. 2011; Li et al. 2015). Some previous studies Ma et al. 2005; Zhang et al. 2015b), as one of the have suggested that CCC can increase plant resis- world’s five major landscaping tree species and tance to salt damage (Liu et al. 2013), UV-B radia- large calibre fast-growing timber species (Olson et tion (Kreslavskii et al. 2011), lodging (Zhang et al. al. 1990; Zuo et al. 2000, 2003; Ju et al. 2007; Cown 2015a) and leaf spot disease (Kundu et al. 2014). et al. 2013); its successful introduction can beau-

Supported by the National Spark Plan Project (No. S2013C100537), College Natural Fund of Jiangsu Province (No. 07KJD210198), Science and Technology Plan Project of Xuzhou (No. XM13B124); Plan Project of Xuzhou Institute of Technology (No. XKY201013)

236 Journal of Forest Science, 66, 2020 (6): 236–243 Original Paper https://doi.org/10.17221/118/2019-JFS tify the environment, enrich biodiversity, improve application would protect coast redwood seed- the local forestry structure, and bring good eco- lings from damaging effects of natural cooling and logical, social and economic benefits. Now, it has drought in autumn and winter. been introduced and cultivated in more than 30 countries, and it has also been successively intro- MATERIAL AND METHODS duced to many provinces in southern China after 1972 (Zuo et al. 2000, 2003; Liu et al. 2006). Coast Site description. The experimental field is lo- redwood was introduced to Xuzhou in 2003 (Zuo cated at a nursery base, Xuzhou Institute of Tech- et al. 2000; Ju et al. 2009). During the Tertiary Pe- nology, in the eastern suburb of Xuzhou, Jiangsu riod various genera of coast redwoods occurred province (34°15'N, 117°11'W), China, belonging throughout the northern hemisphere. Coast red- to the warm temperate semi-humid monsoon cli- wood was eliminated from its former area of distri- mate, with strong spring winds, warm humid sum- bution by the increasingly drier and cooler climates mer and dry cold winter. The sunshine duration of the mid and late Tertiary Period. Temperature is 2 284 to 2 495 hours, while the sunshine rate is and precipitation are the main factors affecting the 52 to 57%, the annual temperature is 14.58 °C, the survival and distribution of coast redwood (Sim- average lowest temperature is –10.52 °C, the aver- mons, Thomas 1975; Ma et al. 2005; Zhang et al. age extreme minimum temperature is –37.43 °C, the 2015b). On average, the mean annual temperature annual accumulated temperature is 5 143.5 °C, the (MAT) for the Sequoia region varies from 13.8 °C average annual frost-free period is about 210 days, to 11.3 °C, the temperature rarely drops below the average annual rainfall is 853.1 mm. The highest –9 °C or rises above 38 °C and the mean mini- average temperature in November is 14 °C and the mum temperature of the coldest month is about lowest average temperature is 4 °C (Figure 1). The 6 °C. Annual precipitation varies between 640 and maximum temperature for the test day is 12 °C and 3 100 mm and mostly winter rains occur in the Se- the minimum temperature is 4 °C. quoia forest region. It is suggested that the coast Plant culture and CCC treatments. The ex- redwood was living in a warm and humid subtrop- periments were carried out during September to ical climate (Ma et al. 2000; Zhang et al. 2015b). November 2014. Uniform 2-years-old seedlings Xuzhou is located in central and eastern China, it of coast redwood were used as experimental mate- belongs to the sub-humid warm temperate con- rial. The experiment consisted of 12 treatments: (1) tinental monsoon climate, four distinct seasons, Single application of CCC (0, 100, 500, 1 000, 1 500, with hot and rainy summer, low temperature and 2 000 mg·l–1) on September 01, 2014 (2014-9-01); drought in winter. According to the above analysis, (2) Two applications of CCC (0, 100, 500, 1 000, the low temperature and drought in autumn and 1 500, 2 000 mg·l–1) on September 01 and Septem- winter are the main stress factors for successful in- ber 08, 2014 (2014-9-01; 2014-9-08). Six CCC solu- troduction of coast redwood to Xuzhou. For years, tions were prepared by dissolving the appropriate studies on adaptability of coast redwood in Xuzhou quantities of 50% CCC water solution in tap water. have shown that low temperature and drought in Spraying with pure water served as a control. Differ- autumn and winter could make the twigs of coast ent concentrations of CCC solutions were sprayed redwood become withered (Ju et al. 2009), which onto plants until drops began to fall from the fo- has limited the cultivation and promotion of coast liage. The experiment was arranged in a complete redwood in central and eastern China. So, improv- randomized design with five replicates for each ing the resistance of coast redwood has become a treatment and each treatment had five seedlings. practical problem that needs to be solved urgently Measurements of growth indices. Growth in- for further application and dissemination. dices, including plant height, crown diameter We could consider the application of a plant and root collar diameter, were measured before growth inhibitor to coast redwoods to limit the CCC application and after the CCC treatment for second growth peak in autumn, increase lignifica- 70 days, computational Equations (1–3): tion of branches, and improve the resistance to low temperature and drought, so that they could safely Increment of plant height: survive in winter in Xuzhou. In the present study, the purpose was to test the possibility that CCC IPH (cm) = H2 – H1 (1)

237 Original Paper Journal of Forest Science, 66, 2020 (6): 236–243

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Figure 1. Agricultural mete- orological indicators: average maximum and minimum temperature (°C), average rainfall (mm) of the month in Xuzhou area, China (2010 to 2018 years)

–

Increment of crown diameter: sity (PPFD) (Januskaitiene 2011; Zhao et al. 2015). Measurements for each leaf were taken three times ICD (cm) = C2 – C1 (2) and averaged, one leaf per seedling and five seedlings randomly selected per treatment. Increment of root collar diameter: Measurements of chlorophyll ﬂuorescence parameters. The third fully expanded leaf counted IRCD (mm) = D2 – D1 (3) from the top of each seedling was selected to test the chlorophyll fluorescence parameters, the actual H , C and D represent the plant height, crown diam- 1 1 1 photochemical quantum yield (Yield) (Genty et al. eter and root collar diameter of coast redwood seedlings 1989 ) and photosynthetic electron transport rate determined on September 01, 2014, respectively. H , C 2 2 (ETR) (Genty et al. 1989; Schreiber 2004) using a and D represent the plant height, crown diameter and 2 chlorophyll spectrometer (MINI-PAM, Walz, Ger- root collar diameter of the corresponding plants deter- many), from 14:00 p.m. to 15:00 p.m. on November mined on November 09, 2014, respectively. 09, 2014. During the measurements, the photosyn- The crown diameter (the mean of the minimum thetically active radiation and temperature in a leaf and maximum canopy diameters) and plant height chamber were set to 1 000 μmol·m–2·s–1 and 10 °C, (the distance between the base of the stem and the respectively. Three measurements per leaf were re- top of the canopy) were measured with a measur- corded and averaged, one leaf per seedling and five ing tape. The root collar diameter (the diameter of randomly selected seedlings per treatment. the base of the plant measured in cm at 5 cm above Statistical analysis. The means ± standard devi- the ground) was measured with a calliper to the ation were calculated using SPSS 19 software (IBM, nearest 1 mm. Armonk, New York, USA). The least significant Measurements of photosynthetic parame- difference (LSD) between the different treatments ters. The measurement of the net photosynthetic was analyzed by the one-way analysis of variance rate (Pn) was performed between 14:00 p.m. and (ANOVA) and the interactions between spraying 15:00 p.m. on November 09, 2014, using a porta- times and concentrations of CCC were analyzed by ble photosynthetic system (LI-6400, LI-Cor 6400, the two-way ANOVA using SPSS 19 software. Cor- USA). Measurements were done on the third fully relations between the measured indicators were expanded leaf counting from the top of each plant analyzed using Origin 8.0 software (OriginLab, –1 at 20 ± 2 °C, 380 ± 15 μmol·mol atmospheric CO2 Northampton, USA). Student’s t-test was applied concentration and 600 µmol·m–2·s–1 saturating to determine the significance of the differences be- light at photosynthetically active photon flux den- tween the individual treatments (P < 0.05 or 0.01).

238 Journal of Forest Science, 66, 2020 (6): 236–243 Original Paper https://doi.org/10.17221/118/2019-JFS

(A) RESULTS

Effects of CCC on the growth of coast redwood seedlings in autumn and winter Figure 2 shows the CCC concentrations increased from 500 to 2 000 mg·l–1, when increments of plant height and crown diameter were significantly lower than those of the control, decreasing gradually with the increase of the concentration, but the reduction was not significant in the range of 1 500 to 2 000 mg·l–1. However, the increment of root collar diameter increased obviously compared to the

0 100 500 1 000 1 500 2 000 control, with a trend of increasing first and then decreasing. The dates of application significantly affected the root collar diameter, followed by the (B) crown diameter, and had no marked effect on the plant height. Two applications significantly increased the root collar diameter compared to single application in a range of 500 to 2 000 mg·l–1, and the maximum was reached at the concentration of 1 500 mg·l–1 with two applications. The results of two-way ANOVA revealed an obvious interaction between the concentration and dates of CCC application that affected the increments of plant height, crown diameter and root collar diameter (F:31.733, P < 0.01; F:57.419, P < 0.01; F:57.311, P < 0.01, respectively). The increments of plant 0 100 500 1 000 1 500 2 000 height and crown diameter negatively correlated with the increment of root collar diameter (p < 0.05) while the increment of plant height positively (C) correlated with the increment of crown diameter (p < 0.01) (Table 1).

Effects of CCC on Pn of coast redwood seedlings in autumn and winter Figure 3 shows that the application of CCC could

help the coast redwood seedlings keep higher Pn than that of the control, with a trend of increasing first and then decreasing with the increase of CCC concentration, then the maximum was reached at 1 000–2 000 mg·l–1 CCC. Two applications were better than single application of 100 mg·l–1 0 100 500 1 000 1 500 2 000 CCC, but two applications significantly decreased CCC concentration (mg·l–1) Pn compared with that of single application of spraying 1x spraying 2x 2 000 mg·l–1 CCC. In coast redwood seedlings –1 Figure 2. Effects of chlorocholine chloride (CCC) on treated with CCC in a range of 500–1 500 mg·l , the increment of plant height (A), increment of crown differences in Pn in leaves sprayed once or twice diameter (B), increment of root collar diameter (C) in were not significant under the same concentration S. sempervirens seedlings under natural cooling and conditions. The two-way ANOVA results showed drought; significant differences at P < 0.05 are shown by an evident interaction between CCC concentra- different letters; n = 5 tions and application dates that affected the Pn of 239 Original Paper Journal of Forest Science, 66, 2020 (6): 236–243

https://doi.org/10.17221/118/2019-JFS

Table 1. Correlation coefficients between indicators determined in this experiment

–2 –1 IPH (cm) ICD (cm) IRCD (mm) Pn (μmol CO2 m ·s ) Yield ICD (cm) 0.903** IRCD (mm) –0.439* –0.435* –2 –1 Pn (μmol CO2 m ·s ) –0.844** –0.784** 0.746** Yield –0.618** –0.644** 0.446* 0.652** ETR –0.214 –0.374* 0.624** 0.492** 0.579**

IPH – increment of plant height; ICD – increment of crown diameter; IRCD – increment of root collar diameter; Pn – net photosynthetic rate; Yield – actual photochemical quantum yield; ETR – photosynthetic electron transport rate; *indicates significance at 0.05 level, ** indicates significance at 0.01 level coast redwood seedlings under natural cooling and tion, the effect difference between two applications drought (F:167.648, p < 0.01). The correlation anal- and single application was not distinct either. ETR ysis indicated that the increment of root collar di- also had a similar effect on Yield. Yield reached the –1 ameter positively correlated with Pn (P < 0.01), and maximum at concentrations of 500–2 000 mg·l the increments of plant height and crown diameter CCC with single application while it reached the –1 negatively correlated with Pn (P < 0.01) (Table 1). maximum at concentrations of 100–2 000 mg·l CCC with two applications. ETR reached the maxi- Effects of CCC on fuorescence parameters mum at concentrations of 1 000–2 000 mg·l–1 CCC of coast redwood seedlings in autumn and winter with single application and reached the maximum Data (Figure 4) document that the application at concentrations of 500–1 500 mg·l– 1 CCC with of CCC could maintain higher Yield and ETR in two applications. Two-way ANOVA results indi- the leaves of coast redwood in autumn and winter. cated an interaction between the CCC concentra- With the increasing concentrations of CCC, Yield tions and application dates that affected Yield and and ETR showed a trend of increasing first and then ETR in the leaves of coast redwood seedlings under decreasing. For Yield, the effect difference between natural cooling and drought. The correlation anal- different concentrations of CCC (500–2 000 mg·l–1) ysis indicated that Yield and ETR were positively was not obvious, and under the same concentra- correlated with Pn (P < 0.01).

DISCUSSION

CCC is a plant growth inhibitor which can change )

–1 vegetative growth of plants, reduce plant height ·s

–2 and increase stem diameter (Wu et al. 2012, 2014), m

2 regulate physiological activity in plants, especially under environmental stress (Kreslavskii et al. 2011; Karimi et al. 2014). Our findings showed a similar

(μmol CO conclusion that the application of CCC signifi- n P cantly decreased the increment of plant height and crown diameter of coast redwood seedlings, and increased the root collar diameter. This study also 0 100 500 1 000 1 500 2 000 showed under the conditions of low temperature CCC concentration (mg·l–1) and drought in autumn and winter in Xuzhou that spraying 1x spraying 2x the high photosynthesis and fluorescence activity were maintained by the application of CCC, which Figure 3. Effect of chlorocholine chloride (CCC) on the indicated that CCC could improve the resistance of net photosynthetic rate (Pn) in S. sempervirens seedlings coast redwood seedlings. The effect of CCC treat- under natural cooling and drought; significant differences ment depended on the concentration and applica- at P < 0.05 are shown by different letters; n = 5 tion dates (Wu et al. 2012; Karimi et al. 2014; Li et

240 Journal of Forest Science, 66, 2020 (6): 236–243 Original Paper https://doi.org/10.17221/118/2019-JFS

(A) portant indicator to evaluate plant resistance (Hu et al. 2016a, b; Polishchuk et al. 2016; Sun et al.

2016). Higher Pn indicates the more organic mat- ter is synthesized in plants and the more energy for consumption, the stronger the ability to resist environmental stress. In this study, the correlation

analysis showed that Pn was significantly positively Yield correlated with the root collar diameter, Yield and ETR (P < 0.01) were significantly negatively correlated with the plant height and plant width (P <

0.01) (Table 1). So Pn could be an important index to evaluate the effect of CCC on alleviating the impact of the low temperature and drought stress. 0 100 500 1 000 1 500 2 000 Our results also show that the application of CCC

(B) could increase Pn compared to the control (Figure 3), which indicated that CCC application could improve the resistance of coast redwood, relieve the stress of low temperature and drought in autumn and winter. The results concur with the findings of Kreslavskii et al. (2011), who also reported that the

application of CCC increased the UV-radiation re-

ETR sistance of common bean according to photosynthetic characteristics improved. Xu et al. (2011) and Dong et al. (2012) also reported that spraying

with CCC increased Pn in the leaves of Ginkgo biloba and Pistacia chinensis. The absorption and the conversion of light ener- 0 100 500 1 000 1 500 2 000 gy can impact photosynthetic efficiency in plants. CCC concentration (mg·l–1) Yield and ETR, as the important fluorescence pa- spraying 1x spraying 2x rameters, reflect the actual light energy conversion efficiency and actual photosynthetic electron trans- Figure 4. Effects of chlorocholine chloride (CCC) on port rate of a plant (Genty et al. 1989; Schreiber the actual photochemical quantum yield of PSII pho- tochemistry (Yield) (A), photosynthetic electron trans- 2004). ETR and Yield are widely used to evaluate the port rate (ETR) (B) in S. sempervirens seedlings under effect of environmental stress on plants, including natural cooling and drought; significant differences at low temperature and drought (Zhang et al. 2015b). P < 0.05 are shown by different letters; n = 5 Studies have shown that drought and low temperature significantly reduced Yield and ETR. Plant al. 2015). In this study, the highest photosynthetic growth inhibitors can improve plant resistance to activity was the main index, and the relatively high low temperature and drought, maintain high Yield growth index (root collar diameter, plant height and ETR (Li et al. 2015; Zhang et al. 2015a; Hu et al. and crown diameter) was used a reference to deter- 2016a, b). Our findings showed that Yield and ETR mine the appropriate treatment mode of CCC. So, increased significantly in the leaves of coast red- the application effect of 1 000–1 500 mg·l–1 CCC wood seedlings treated with CCC (Figure 4), which was the best. indicated that the application of CCC increased the Photosynthesis converts the light energy into resistance of coast redwood seedlings. chemical energy which is the basis of plant survival (Bernacchi et al. 2013). Some findings suggested CONCLUSION that photosynthetic capacity decreased under the environmental stress. The photosynthetic rate (Pn) CCC application onto coast redwood seedlings is the most important parameter to evaluate pho- inhibited the elongation of their shoots, improved tosynthetic capacity of plants. So, Pn is also an im- their Pn, Yield and ETR, enhanced their resistance 241 Original Paper Journal of Forest Science, 66, 2020 (6): 236–243

https://doi.org/10.17221/118/2019-JFS and alleviated the stress caused by low tempera- Karimi M., Ahmadi A., Hashemi J., Abbasi A., Angelini L.G. tures and drought in the autumn and winter. The (2014): Effect of two plant growth retardants on steviol best protection was obtained in the plants treated glycosides content and antioxidant capacity in Stevia (Ste- with CCC at 1 000–2 000 mg·l−1. Therefore, the via rebaudiana Bertoni). Acta Physiologiae Plantarum, application of CCC in early September could be a 36: 1211–1219. suitable strategy for coast redwood cultivated and Kreslavskii V.D., Lubimov V.Y, Kotova L.M., Kotov A.A. promoted in the Xuzhou region, China. However, (2011): Effect of common bean seedling pretreatment the experiment was carried out from September to with chlorocholine chloride on photosystem II tolerance November, and the damage by severe low tempera- to UV-B radiation, phytohormone content, and hydrogen ture and drought from December to February to peroxide content. Russian Journal of Plant Physiology, 58: the coast redwood and the mitigative effect of CCC 324–329. should be further studied. Kundu S., Dey A., Bandyopadhyay A. (2014): Chlorocholine chloride mediated resistance mechanism and protection References against leaf spot disease of Stevia rebaudiana Bertoni. European Journal of Plant Pathology, 139: 511–524. Bernacchi C.J., Bagley J.E., Serbin S.P., Ruiz-Vera U.M., Li Y., He B.H., Huang X.H., Mao W.T., Yu C., Qin H.J. (2015):

Rosenthal D.M., Vanloocke A. (2013): Modelling C3 pho- Response of chlorophyll and chlorophyll fluorescence tosynthesis from the chloroplast to the ecosystem. Plant, parameters of Scaevola aemula ‘Sunfan’ to chlorocholine Cell & Environment, 36: 1641–1657. chloride. Journal of Southwest University, 37: 20–27. (in Cown D., Marshall H., Silcock P., Meason D. (2013): Sawn Chinese). timber grade recovery from a planted coast redwood stand Liu C., Xia X., Yin W., Huang L., Zhou J. (2006): Shoot re- growing in New Zealand. New Zealand Journal of Forestry generation and somatic embryogenesis from needles of Science, 43: 1–11. redwood (Sequoia sempervirens (D.Don.) Endl.). Plant Cell Dong Q., Wang J., Pang M., Feng X.B., Bai Z.Y., Lu B.S. (2012): Reports, 25: 621–628. Effects of growth regulators on photosynthetic and physi- Liu J.X., Wang R.L., Wang T., Hu Y. (2013): Effect on seedlings ological indices and chlorophyll fluorescence parameters growth of Pinus yunnanensis Franch. after seed soaking of Pistacia chinensis. Acta Botanica Boreal, 32: 484–490. with chlormequat chloride in the high NaCl stress. Hubei Januskaitiene I. (2011): Effects of substrate acidity and UV-B Agricultural Science, 52: 3582–3585. (in Chinese) radiation on photosynthesis of radishes. Central European Ma Q.W., Li F.L., Li C.S. (2005): The coast redwoods (Sequoia, Journal of Biology, 6: 624–631. Taxodiaceae) from the Eocene of Heilongjiang and the Ju S.M., Gao M.X., Xu D.L. (2007): Research on the cutting Miocene of Yunnan, China. Review of Palaeobotany and for Sequoia sempervirens. Journal of Xuzhou Institute of Palynology, 135: 117–129. Technology, 22: 40–43. (in Chinese). Olson D.F., Roy D.F., Walters G.A. (1990): Sequoia semper- Ju S.M., Gao M.X., Xu D.L. (2009): Study on the asexual rapid virens (D. Don) Endl. In: Barnes R.M., Honkala B.H. (eds): propagation of cold-resistant Sequoia sempervirens. Practi- Silvics of North America. Agriculture Handbook 654, Vol. 1 cal Forestry Technology, 1: 23–27. (in Chinese). Conifers. Washington, DC, USDA Forest Service: 541–551. Hu H.Q., Wang L.H., Li Y.L., Sun J.W., Zhou Q., Huang X.H. Polishchuk O.V., Vodka M.V., Belyavskaya N.A., Khomochkin (2016a): Insight into mechanism of lanthanum (III) induced A.P., Zolotareva E.K. (2016): The effect of acid rain on ultra- damage to plant photosynthesis. Ecotoxicology and Envi- structure and functional parameters of photosynthetic ap- ronmental Safety, 127: 43–50. paratus in pea leaves. Cell and Tissue Biology, 10: 250–257. Hu H.Q., Wang L.H., Zhou. Q., Huang X.H. (2016b): Com- Schreiber U. (2004): Pulse-Amplitude-Modulation (PAM) bined effects of simulated acid rain and lanthanum chlo- fluorometry and saturation pulse method: an overview. ride on chloroplast structure and functional elements in In: Papageorgiou G.C., Govindjee (eds): Chlorophyll a rice. Environmental Science and Pollution Research, 23: Fluorescence: a Signature of Photosynthesis. Dotrecht, 8902–8916. Springer: 279–319. Genty B., Briantais J.M., Baker N.R. (1989): The relationship Simmons I.G., Thomas R.V.M.A. (1975): Conservation of the between the quantum yield of photosynthetic electron California coast redwood and its environment. Environ- transport and quenching of chlorophyll fluorescence. mental Conservation, 2: 29–38. Available at https://doi. Biochimica et Biophysica Acta (BBA) – General Subjects, org/10.1017/S0376892900000618 990: 87–92. Sun J., Hu H., Li Y., Wang L., Zhou Q., Huang X. (2016): Ef- fects and mechanism of acid rain on plant chloroplast ATP

242 Journal of Forest Science, 66, 2020 (6): 236–243 Original Paper https://doi.org/10.17221/118/2019-JFS

synthase. Environmental Science and Pollution Research, character, photosynthetic and material production of sweet 23: 18296–18306. sorghum. Southwest China Journal of Agricultural Science, Wang H.Q., Li H.S., Liu F.L., Xiao L.T. (2009): Chlorocholine 28: 1972–1976. (in Chinese) chloride application effects on photosynthetic capacity and Zhang J.W., D’Rozario A., Adams J.M., Li Y., Liang X.Q., photoassimilates partitioning in potato (Solanum tubero- Jacques F.M. (2015b): Sequoia maguanensis, a new Miocene sum L.). Scientia Horticulturae, 119: 113–116. relative of the coast redwood, Sequoia sempervirens, from Wang H.Q., Xiao L.T., Tong J.H., Liu F.L. (2011): Foliar ap- China: Implications for paleogeography and paleoclimate. plication of chlorocholine chloride improves leaf mineral American Journal of Botany, 102: 103–118. nutrition, antioxidant enzyme activity, and tuber yield of Zhao X., Li Y., Zheng M., Bian X., Liu M., Sun Y., et al. (2015): potato (Solanum tuberosum L.). Scientia Horticulturae, Comparative analysis of growth and photosynthetic char- 125: 521–523. acteristics of (Populus simonii × P. nigra) × (P. nigra × P. Wu M.X., Zhang S.S., Jiang X.M., Yu X.H., Liu Y., Che X. simonii) hybrid clones of different ploidides. PLoS ONE, (2014): Effects of CCC treatment on vernalization-key 10: e0119259. enzyme activity in broccoli. Journal of Zhejiang University Zuo X., Bai S., Shao J., Peng M., Qi R., Wang Y. (2003): (Science Edition), 41: 63–66. Growth of Sequoia sempervirens introduced to Yunnan Wu R.H., Li Y., Wang S., Niu X.H., Liu P. (2012) Effect of and reforestation prospect. Yunnan Forestry Science and plant growth retardants on the growth and development Technology 104: 2–10. (in Chinese) of potted rose. Acta Botanica Boreal, 32: 767–773. Zuo X., Qi R., Wang Y., Shao J., Peng M. (2000): Introduction Xu F., Zhang W.W., Sun N.N., Li L.L., Cheng S.Y., Wang Y. and ecological adaptability of Sequoia sempervirens Endl. (2011) Effects of chlorocholine chloride on photosynthesis in China. Yunnan Forestry Science and Technology, 93: metabolism and terpene trilactones biosynthesis in the leaf 36–40. (in Chinese) of Ginkgo biloba. Acta Horticulturae Sinica, 38: 2253–2260. Received: October 6, 2019 Zhang F., Wang Y.Q., Zhu K., Zhang Z.P., Zou J.Q. (2015a): Accepted: May 22, 2020 Effect of chlormequat chloride application on lodging

243 Original Paper Journal of Forest Science, 66, 2020 (6): 244–251

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Morphological and molecular identification of ectomycorrhizal fungi associated with Persian oak (Quercus brantii Lindl.) tree

Behnaz Yousefshahi1, Masoud Bazgir2*, Samad Jamali3, Fatemeh Valizadeh Kakhki2

1Department of Soil Science, Faculty of Agriculture, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran 2Department of Soil and Water Engineering, Faculty of Agriculture, Ilam University, Ilam, Iran 3Department of Plant Protection, Faculty of Agriculture, Razi University, Kermanshah, Iran *Corresponding author: [email protected]

Citation: Yousefshahi B., Bazgir M., Jamali S., Kakhki F.V. (2020): Morphological and molecular identification of ectomycorrhizal fungi associated with Persian oak (Quercus brantii Lindl.) tree. J. For. Sci., 66: 244–251.

Abstract: Identification of ectomycorrhizal (ECM) fungi in different ecosystems has major significance. In this research, to identity ECM fungi, we used two methods including the morphological method and the molecular method that is more precise. Basidiocarp collection of fungi associated with oak tree (Quercus brantii Lindl.) roots was carried out in the spring season 2016 and was identified by morphological and molecular methods. We also checked macroscopic and microscopic features and measured each structure using BioloMICS Measures software. To verify the morphological identification, the internal transcribed spacer (ITS) region was amplified by PCR using the primer pair ITS1/ITS4, and the sequences were analyzed. According to the morphological observations, the identified species were Amanita crocea, Boletus comptus, Tricholoma giganteum, and Inocybe rimosa. Besides, based on molecular techniques by comparing sequences, we identified five species out of the eight ones as A. crocea and other species as T. giganteum, I. rimosa and B. comptus. Both morphological and molecular methods are necessary for identifying ECM fungi associated with tree roots in the Zagros zone in the west of Iran.

Keywords: mycorrhizal symbiosis; forest ecosystem; Amanita; Tricholoma; Inocybe; Boletus

In recent decades, oak tree decline has been a wide- fungi called ectomycorrhiza in the soil, which brings spread problem in the Zagros zone in the west of Iran. benefits for them (Brundrett 2009; Itoo, Reshi 2013; Different conditions lead to the decline, including Wurzburger et al. 2017). Numerous studies have indi- drought, soil nutrient depletion, attack of pests and cated that the mycorrhiza can promote plant growth diseases, and dust storm (Sagheb-Talebi et al. 2014). and survival by increasing their tolerance to biotic Mycorrhizal symbiosis is especially ectomycorrhizal and abiotic stresses (Sylvia, Williams 1992; Wu 2017). by increasing drought resistance and enhancing the ECM are commonly found in temperate forests or ability of plants to uptake nutrient elements, decreas- in taiga, where other fungi such as truffles, boletes, ing tree decline and mortality (Brady, Weil 2008). amanitas, and chanterelles exist. They form a sym- In the forest, there is an obligatory symbiotic re- biosis with tree roots including oak, pine, birch, eu- lationship between tree roots and a certain group of calyptus, and European spruce (Querejeta et al. 2014;

Supported by the Ilam University, Project No. 1396-448A

244 Journal of Forest Science, 66, 2020 (6): 244–251 Original Paper https://doi.org/10.17221/31/2020-JFS

Korhonen et al. 2019). ECM have an essential role from 7 to 7.8. Fungal samples were collected during in the forest growth and health by improving the the growing season in May 2016. Fruit bodies were uptake of nutrients such as phosphorus, nitrogen, collected within a distance of 2–3 m from the tree and micronutrient elements from the soil (Smith, trunk on the soil surface. Read 2008). Mirzaei and Heydari (2014) studied the Morphological identification. We investigated relationship between environmental factors, colo- morphological features including the shape and nization and abundance of arbuscular mycorrhizal dimensions of the pileus and stipe, the presence fungi associated with Amygdalus scoparia in Zagros or absence of the volva, gill attachment, the spore forests. They identified 7 species of AMF including print colour, clamp connection, shape and size of Glomus fasciculatum, G. intraradices, G. mosseae, basidiospores, shape, size and position of cystidia, G. claroideum, G. drummondi, G. caledonium and shape and number of stigmata. Specimens were Gigaspora gigantea through morphological charac- identified based on available taxonomic keys and teristics in the region. publications (Moser, Kibby 1983; Knudsen, Vester- Molecular techniques and the DNA sequencing holt 2008). After the preliminary investigation, the analysis can be used as faster and more accurate specimens were dried using a dehydrator. methods for the identification of ECM with high Molecular identification. Doyle and Doyle morphological diversity. In many studies, molecu- (1990) method with slight modification was used lar techniques have been used to identify ECM (e.g. to extract the total DNA from 5 mg of fresh basidi- Larsson et al. 2009; Jairus et al. 2011; Bahram et al. ocarps. The fungal tissue (fruit body) and the ex- 2013; Sebastiana et al. 2013; Lothamer et al. 2014; tracted DNA were transferred to Eppendorf tubes. Pushpa et al. 2014; Vizzini et al. 2014; Latha, Mani- The samples were then placed in a mortar contain- mohan 2016). Many researchers have used the mark- ing liquid nitrogen and powdered using a porce- ers to identify the genus and species of ECM (White lain pestle. The fungal tissue was then suspended et al. 1990; Pushpa et al. 2014). In Iran, the surface in 700 ml extraction buffer consisting of 100 mM area of forest land (% of land area) was 6.6% in 2016. Tris-HCl (pH 9.0), 0.5 M EDTA (pH 8.0), 1.4 M So, it is essential to identify ectomycorrhizal sym- NaCl, 2% hexadecyltrimethylammonium bromide biosis fungi in these ecosystems. Therefore, the aim (CTAB), 0.2% mercaptoethanol and centrifuged for of this research is to provide a morphological and 30 min at 65°C. The tubes containing the samples molecular characterization of ECM that increase were inverted every 10 min, and 700 μl chloroform/ drought resistance via a symbiotic relationship be- isoamyl alcohol (24:1, v/v) was added to the tubes. tween oak tree roots and ECM. The tubes were centrifuged for 20 min at 14 000 g for the phases to be separated. Then, the superna- MATERIAL AND METHODS tant was removed and 2 μl of RNase A was added to the aqueous phase which was incubated at 37 °C Site description. The geographical location of the for 30 min. Seventy μl of sodium acetate 2.5 M (or studied area is between longitude 46° 41' 23.76'' E potassium acetate) and 400 μl isopropanol which and latitude 33° 19' 28.95'' N, with a total area of had been stored for 30 min–24 h at –20 °C was about 4 417 hectares. The elevation range varies added to each tube. The solution was centrifuged at from 1 350 m to 1 756 m a.s,l. with slopes between 14 000g for 10 min. for developing the DNA pellet. 10 and 15% and north-south facing aspect. The The upper phase was discarded and washed away major vegetation cover is composed of Persian oak with 70% (v/v) ethanol, and centrifuged for 60 sec- (Quercus brantii Lindl.) trees which are accom- onds min at 12 000g. The pellet containing DNA panied by wild pistachio (Pistacia spp.) trees and was solubilized in 100 μl sterile deionized water hawthorn (Crataegus pontica C. Koch) trees (Sa- and kept at 4 °C for 24 h. Finally, the tubes were gheb-Talebi et al. 2014). The average age of forest stored at –20 °C until further use. The quality of trees is over 150 years. According to the De Mar- the extracted DNA was determined by electropho- tonne climatic classification, the climate is temper- resis on 0.8% agarose gel, followed by staining using ate semi-humid with mean annual precipitation of ethidium bromide. In the last analysis, the gel was 592.5 mm and mean annual temperature of 19.4 °C. exposed to UV radiation. The soils in the study area are classified as Incepti- Amplification of ITS by PCR. Amplification of sols with silty clay loam texture and soil pH ranges the internal transcribed region of nuclear ribosom-

245 Original Paper Journal of Forest Science, 66, 2020 (6): 244–251

https://doi.org/10.17221/31/2020-JFS al DNA (rDNA) was done using universal prim- cation. In 2016, the specimens were retrieved from ers of ITSl and ITS4 with the respective sequenc- Bivereh oak forest in Malekshahi region, Ilam proves of 5’-TCCGTAGGTGAACCTGCGG-3’ and ince (west of Iran). 5’-TCCTCCGCTTATTGATATGC-3’ (White et al. 1990). The mixture used for PCR included 12.5 μl Morphological identification of Master Mix (2X), 1 μl from each of the prim- In the present study, four species of ectomycor- ers mentioned above (100 pmol/µl), 4 µl of diluted rhizal fungi (ECM) associated with oak trees were DNA sample (100 pmol/µl) and 6.5 µl of sterile de- identified which are described as follows: ionized water for the total 25μl reaction volume. The PCR program used for amplification consisted Amanita crocea (Quel.) Singer of 3 min. of initial denaturation at 95°C, denatur- A. crocea has a cap of 4–10 cm in size, convex, ation and annealing for 35 cycles at 94 °C and 60 °C saffron orange colour in the centre and brown in and for 60 seconds, respectively and 72 °C of exten- the margin. The gills are free and cream in mass. sion for 90 seconds. The final extension stage had Stipe 90–120 × 15–25 mm, cream to pale orange, duration of 5 min. at 72 °C. To test whether there without annulus, almost equal and has a volva. are any contaminations by reagents and reaction Lamellae cream-colored and free. Spores hyaline, buffers, every amplification set contained DNA- smooth, globose to subglobose and 8–12 × 7–9.5 μm. free control samples. The quality of the extracted Spore print white (Figure 1; A, B). DNA was determined by electrophoresis on 0.8% agarose gel followed by staining using ethidium Boletus comptus Simonini 1993 bromide (Sambrook et al. 1982) and photographed In this fungus, the cap is convex, 8–10 cm, pinkish under UV light. or greyish pink. The tubes are yellow, pores small, Sequencing of amplified ITS regions. The am- orange-brown, turn black when pressed. The stipe plified segment was purified using a GeneJET PCR is thick, clavate or club-shaped, 7–9 × 3–4 cm, yel- Purification Kit (Fermentas, UK). The sequencing low to brown and without annulus. The stem base was done using the primers ITS1 or ITS4, and the is bulbous, rooted in the base and immersed in the nucleotide sequence comparisons were performed earth. Spores 10–15 × 6–7 μm and spores print are using Blast Multiple Alignment Tool (BLASTn) net- brown. Reaction with Melzer’s reagent was positive work sequences against the National Centre for Bio- (Figure 1; C, D). technology Information (NCBI, http://www.ncbi. nlm.nih.gov/Entrez) (Bethesda, MD, USA) database. Tricholoma giganteum Massee, Bull. Misc. Ihf. All DNA sequences of the ITS regions from fruit Kew 1912 bodies were submitted to the GenBank (NCBI). Tricholoma giganteum has a cap of 10–30 cm in Statistical and phylogenetic analyses. Se- size, the colour is cream, white to greyish-white, quences of the ITS regions were used to study phy- initially convex, then flat and glabrous. Gills grey- logenetic relationships of the studied taxa. Mul- ish-white, sinuate (notched), crowded of several tiple alignments were performed with CLUSTALW lengths and 5–11 cm. The stipe is central, white, (Thompson et al. 1994) and BIOEDIT v.7.0.9 (Hall glabrous, solid, 5–10 × 3–5 cm and without an- 1999) tools were used for multiple alignments and nulus. Spores hyaline, smooth, obviate and 8–10 × manually optimized, respectively. Distance methods 6–7 μm. Spore print white. Cystidia not seen. Hy- were used for phylogenetic analysis. The neighbour- phae with clamps (Figure 1; E, F). joining (NJ) method proposed by Saitou and Nei (1987) was applied in constructing tree topology. Inocybe rimosa (Bull.) Bootstrap resampling was computed 1 000 times for This fungus has a small cap, 2–8 cm; conical with assessing branching confidence (Felsenstein 1985). a bump in the centre and yellow to brown. Gills at- tached to the stem in immature state and away from RESULTS it in mature state and grey to brown. Stipe 50–90 × 7–9 mm, equal, without annulus and whitish to In this research, we tried to recognize fungi grow- pale yellowish. Spores smooth, ellipsoidal and 9–12 ing around trees as ectomycorrhiza and symbiotic × 4.5–7 µm. Spore print is brown. Cheilocystidia fungi with oak trees by their collection and identifi- cylindrical and 30–65 x 10–22 µm (Figure 1; G, H).

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1 ( A ) ( C ) 2 3 ( D ) 4

7 ( B ) 8 a 9

13 ( E ) ( G ) 14

18 ( F ) 19 ( H )

Figure 1. Amanita crocea (A) and its spores (B); Boletus comptus (C) and its spores (D); Tricholoma giganteum (E) and its spores (F); Inocybe rimosa (G) and its spore (H)

Molecular Identification value column shows that there is a very high simi- Comparing the reported sequences (Table 1) larity between our sequences and the sequences in suggested that out of the eight ECM species gath- NCBI. The internal transcribed spacers (ITS1 and ered from various regions in the study area, five ITS2) and the 5.8S ribosomal gene of all specimens were identified as Amanita crocea. Other species were amplified using ITS4 and ITS1 universal were identified as Inocybe rimosa, Tricholoma gi- primers, and a single approximately 600–625 bp ganteum and Boletus comptus. As well as, the e- band was obtained via agarose gel analysis for

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Table 1. The results of DNA sequence analysis of the ITS region based on GenBank BLAST searches

Maximum Base Query BLAST best match to Code EMF taxon name ID Accession ident. pairs used coverage E-value vouchered specimen (%) (number) (%) AmCr1 Amanitaceae MF278764 Amanita crocea 99 600 98% 0.0 AmCr2 Amanitaceae MF278765 Amanita crocea 98 600 97% 0.0 TrGi1 Tricholomataceae MG867660 Tricholoma giganteum 99 600 99% 0.0 AmCr3 Amanitaceae MF278766 Amanita crocea 98 600 98% 0.0 InRi1 Inocybaceae MF278770 Inocybe rimosa 99 625 99% 0.0 AmCr4 Amanitaceae MF278767 Amanita crocea 99 600 99% 0.0 AmCr5 Amanitaceae MF278768 Amanita crocea 99 600 97% 0.0 BoCo1 Boletaceae MF278769 Boletus comptus 99 600 98% 0.0

BLAST – program that finds similar protein or nucleotide sequences to your target sequence, E-value – probability due to chance

each species. The ITS sequences of five speci- posited in GenBank. Using the cladistic meth- mens of Amanita (Accession no.: MF278764 to od, the phylogenetic tree length was 734 with MF278768), one specimen of Tricholoma (Acces- CI = 0.87; RI = 0.94; RCI = 0.82 for all sites and sion no.: MG867660), one specimen of Boletus parsimony-informative sites were iCI = 0.87 and (Accession no.: MF278769) and one specimen iRI = 0.94. According to phylogenetic analysis, of Inocybe (Accession no.: MF278770) from Iran the studied isolates were clustered in a distinct showed 98–99% homology with A. crocea, 99% monophyletic group related to A. crocea, T. gigan- homology with T. giganteum, 99% homology with teum, B. comptus, and I. rimosa from other authors B. comptus and 99% homology with I. rimosa de- (Figure 2).

30 A manita crocea K J638266

60 A manita crocea MF278765 A manita crocea MF278767 60 100 A manita crocea MF278768 100 A manita crocea MF278766

100 A manita crocea MF278764 Tricholoma giganteum JN 192443 Tricholoma giganteum JX 068527 100 Tricholoma giganteum MG867660 100 Tricholoma giganteum JX 193694 Inocybe rimosa MF278770

100 Inocybe rimosa JF908111 100 Inocybe rimosa EU123476 Boletus comptus MF278769 100 Boletus comptus KC734539

1 50 2 Figure 2. Rooted 50% majority rule consensus tree resulting from 1 000 bootstrap replications of the parsimony analysis of the ITS rDNA sequences (Consistency index: CI = 0.87; Retention index: RI = 0.94; Rescaled consistency index: RCI = 0.82). The analysis was conducted using the heuristic search algorithm. Numbers on the branches are the bootstrap values (%). The red triangles refer to our specimens in Iran

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DISCUSSION acteristics of this fungus, Kim et al. (1998) gathered similar results. The genus Inocybe and the species In this paper, we recognized four agaric fungi as- I. rimosa (Figure 1; F) are commonly found in rich sociated with oak trees in Zagros forest according soils, and often on limey soils. These fungi have an to morphological and molecular characteristics. ectomycorrhizal symbiosis with a wide range of The correct identification of the fungi, especially host trees, especially conifers and oak, alder, etc. agaric fungi, is not always easy (Diez et al. 2002). (Jacobsson 2008; Kirk et al. 2008). The genus Ino- Identification of ectomycorrhizal (ECM) fungi is cybe is not usually suitable for eating. The results traditionally carried out with the help of morpho- of morphological identification are in line with the logical characteristics (Amicucci et al. 1998) or by findings of other researchers (Cripps 1997). biochemical, olfactory, biophysical, and immuno- Temperate broadleaved forests are the habitat logical techniques (Papa et al. 1987; Zambonelli et of Boletus comptus (Figure 1; G) which has an ec- al. 1993; Gandeboeuf et al. 1994; Neuner-Plattner tomycorrhizal symbiosis with oak trees (Quercus) et al. 1999). According to the studies mentioned (Estadès et al. 2004). This fungus found in Europe before, very similar morphological features and the and in the Mediterranean region such as Italy, requirement of biological material using accurate- Montenegro and Spain is not edible either. The re- ly identified mycorrhizal fungi is limited (Jamali, sults of morphological identification are consistent Banihashemi 2013). with some previous studies (Simonini 1992; Simo- In recent decades, one useful method for precise nini 1998). identification, taxonomic and phylogenetic studies In summary, we applied morphotyping and mo- of ECM has been the amplification of the internal lecular methods to characterize ECM fungi associ- transcribed spacer (ITS) and the intergenic spacer ated with oak trees and four ECM including I. ri- (IGS) (Henrion et al. 1994; Paolocci et al. 1999). mosa, B. comptus, A. crocea and T. giganteum were In our molecular study, each specimen was ampli- identified which are suitable for mycorrhizal sym- fied using the primer pair ITS1 and ITS4, and an biosis with the ecological characteristics of Iranian amplicon of about 650–660 bp was obtained for oak trees in the Bivareh habitat. However, with most specimens which was in accordance with others more than 1.8 million hectares of forest coverage (Diez et al. 2002; Ferdman et al. 2005). Based on the with different tree species in the Zagros zone, there ITS region sequences, these specimens showed 98– is many more ECM that remained to characterize. 99% homology with authentic specimens deposited Moreover, according to the results, the molecular in GenBank. The accuracy of species identification technique was more precise than the morphologi- was confirmed by phylogenetic analysis. The genus cal method because it characterized other four spe- Amanita (Figure 1; E) has an ectomycorrhizal sym- cies. Therefore, the identification of ECM fungi by biosis with a variety of host plants, especially oak, both methods in these ecosystems is crucial and it beech, and conifers. It is widely found in North should be noted that in poor soils and when there is America and Europe (Pande et al. 2004). This fun- water shortage due to the low rainfall, the presence gus was previously identified in Iran and the au- of ECM increases soil fertility and water availability. thors reported the morphological characteristics of A. crocea and several other species of this genus and achieved similar results (Bahram et al. 2006). References The results of molecular identification for one of the samples led to the identification of T. giganteum. Amicucci A., Zambonelli A., Giomaro G., Potenza L., Stocchi In a similar study with molecular sequencing, the V. (1998): Identification of ectomycorrhizal fungi of the identification of this genus and species was carried genus Tuber by species specific ITS primers. Molecular out using basidiocarps and pure culture of fungi Ecology, 7: 273–277. and the authors obtained similar results (Pushpa et Bahram M., Asef M.R., Zarre S.H., Abbasi M., Reidl S. al. 2014). The individuals of the genus Tricholoma (2006): Addition to the knowledge of Amanita (Agaricales, (Figure 1; H) are usually ECM that have a symbiotic Pluteaceae) from Iran. Rostaniha, 7: 107–119. (in Persian) relationship with different species of broadleaved Bahram M., Kõljalg U., Kohout P., Mirshahvaladi S., Tedersoo and coniferous trees. Tricholoma giganteum is ed- L. (2013): Ectomycorrhizal fungi of exotic pine plantations ible. In a study regarding the morphological char- in relation to native host trees in Iran: evidence of host

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https://doi.org/10.17221/31/2020-JFS

range expansion by local symbionts to distantly related in Korea (I) – Morphological charateristics of fruitbody host taxa. Mycorrhiza, 23: 11–19. and environmental condition in habitat of T. giganteum. Brady N.C., Weil R.R. (2008): The Nature and Properties of The Korean Journal of Mycology, 26: 182–186. Soils. (ed.) New Jersey, Prentice-Hall: 472–477. Kirk P., Cannon P.F., Minter D.W., Stalpers J.A. (2008): Ains- Brundrett M.C. (2009): Mycorrhizal associations and other worth and Bisby’s Dictionary of the Fungi. In: Lang E.J.: means of nutrition of vascular plants: understanding the Edible fungi. Wallingford, CAB International: 230–321. global diversity of host plants by resolving conflicting Knudsen H., Vesterholt J. (2008): Funga Nordica: Agaricoid, information and developing reliable means of diagnosis. Boletoid and Cyphelloid Genera. In: Knudsen H., Vester- Plant and Soil, 320: 37–77. holt J. (eds): Fungi. Copenhagen, Nordsvamp: 450–471. Cripps C.L. (1997): The genus Inocybe in Montana aspen Korhonen A., Lehto T., Heinonen J., Repo T. (2019): Whole- stands. Mycologia, 89: 670–688. plant frost hardiness of mycorrhizal (Hebeloma sp. or Díez J., Manjón J.L., Martin F. (2002): Molecular phylogeny Suillus luteus) and non-mycorrhizal Scots pine seedlings. of the mycorrhizal desert truffles (Terfezia and Tirma- Tree Physiology, 39: 951–960. nia), host specificity and edaphic tolerance. Mycologia, Larsson E., Ryberg M., Moreau P.A., Mathiesen Å.D., Jacobs- 94: 247–259. son S. (2009): Taxonomy and evolutionary relationships Doyle J.J., Doyle J.L. (1990): Isolation ofplant DNA from within species of section Rimosae (Inocybe) based on ITS, fresh tissue. Focus, 12: 39–40. LSU and mtSSU sequence data. Persoonia, 23: 86–98. Estadès A., Lannoy G. (2004): Les bolets européens. Bulletin Latha K.D., Manimohan P. (2016): Five new species of Mycologique et Botanique Dauphiné-Savoie 44: 3–79. Inocybe (Agaricales) from tropical India. Mycologia, Felsenstein J. (1985): Phylogenies and the comparative 108: 110–122. method. The American Naturalist, 125: 1–15. Lothamer K., Brown S.P., Mattox J.D., Jumpponen A. (2014): Ferdman, Y., Aviram S., Nurit R.B., Trappe J.M., Kagan-Zur Comparison of root-associated communities of native and V. (2005): Phylogenetic studies of Terfezia pfeilii and Choi- non-native ectomycorrhizal hosts in an urban landscape. romyces echinulatus (Pezizales) support new genera for Mycorrhiza, 24: 267–280. southern African truffles: Kalaharituber and Eremiomyces. Mirzaei J., Heydari M. (2014): Relationship between en- Mycological Research, 109: 237–245. vironmental factors, colonization and abundance of Gandebœuf D., Dupré C., Chevalier G. (1994): Différencia- arbuscular mycorrhizal fungi associated with Amygdalus tion des truffes européennes par l’analyse des isoenzymes. scoparia in Zagros forests. Iranian of Journal of Forest, Acta Botanica Gallica, 141: 455–463. 6: 445–456. Hall T.A. (1999): BioEdit: a user-friendly biological sequence Moser M., Kibby G. (1983): Keys to agarics and boleti. In: alignment editor and analysis program for Windows. Nu- Polyporales, Boletales, Agaricales, Russulales. London, cleic Acids Symposium Series, 95: 95–98. Roger Phillips: 252–266. Henrion B., Chevalier G., Martin F. (1994): Typing truffle Neuner-Plattner I., Grabher T., Hall I.R., Stöffler G., Griffin species by PCR amplification of the ribosomal DNA spac- F., Haselwandter K. (1999): A comparison of immunologi- ers. Mycological Research, 98: 37–43. cal assays for the identification of Tuber spp. and other Itoo Z.A., Reshi Z.A. (2013): The multifunctional role of ec- edible ectomycorrhizal fungi. Mycological Research, tomycorrhizal associations in forest ecosystem processes. 103: 403–412. The Botanical Review, 79: 371–400. Pande V., Palni U.T., Singh S.P. (2004): Species diversity of Jacobsson S. (2008): Key to Inocybe. In: Knudsen H., Vest- ectomycorrhizal fungi associated with temperate forest erholt J. (eds): Funga Nordica. Agaricoid, boletoid and of Western Himalaya: a preliminary assessment. Current cyphelloid genera. Copenhagen, Nordsvamp: 868–906. Science, 21: 1619–1623. Jairus T., Mpumba R., Chinoya S., Tedersoo L. (2011): Inva- Paolocci F., Rubini A., Granetti B., Arcioni S. (1999): Rapid sion potential and host shifts of Australian and African molecular approach for a reliable identification of Tuber ectomycorrhizal fungi in mixed eucalypt plantations. New spp. ectomycorrhizae. FEMS Microbiology Ecology, 28: Phytologist, 192: 179–187. 23–30. Jamali S., Banihashemi Z. (2013): Species-specific ITS prim- Papa G., Balbi P., Audisio G. (1987): Preliminary study of ers for the identification of Picoa juniperi and Picoa lefe- fungal spores by pyrolysis-gas chromatography. Journal bvrei and using nested-PCR for detection of P. juniperiin of Analytical and Applied Pyrolysis, 11: 539–548. planta. Molecular Biology Reports, 40: 5701–5712. Pushpa H., Purushothama K.B., Ramesh-Thirumalesh D.H. Kim H.K., Kim Y.S., Seok S.J., Kim G.P., Cha D.Y. (1998): (2014): Taxonomic studies and molecular characterisation Artificial cultivation of Tricholoma giganteum collected of Tricholoma giganteum and Calocybe indica isolates

250 Journal of Forest Science, 66, 2020 (6): 244–251 Original Paper https://doi.org/10.17221/31/2020-JFS

from Bangalore. Journal of Biochemical Technology, 3: Sylvia D.M., Williams S.E. (1992): Vesicular-arbuscular 218–220. mycorrhizae and environmental stress. Mycorrhizae in Querejeta J., Egerton-Warburton L.M., Allen M.F. (2009): Sustainable Agriculture, 54: 101–124. Topographic position modulates the mycorrhizal response Thompson J.D., Higgins D.G., Gibson T.J. (1994): CLUSTAL of oak trees to interannual rainfall variability. Ecology, 90: W: improving the sensitivity of progressive multiple se- 649–662. quence alignment through sequence weighting, position- Saitou N., Nei M. (1987). The neighbor-joining method: a new specific gap penalties and weight matrix choice. Nucleic method for reconstructing phylogenetic trees. Molecular Acids Research, 22: 4673–4680. Biology and Evolution, 4: 406–425. Vizzini A., Simonini G., Ercole E., Voyron S. (2014): Boletus Sambrook J., Fritsch F.E., Maniatis T. (1982): Molecular Clon- mendax, a new species of Boletus sect. Luridi from Italy ing: a Laboratory Manual (Vol. 545). In: Janssen K. (ed.): and insights on the B. luridus complex. Mycological Pro- Isolation of High-Molecular-Weight DNA from Mam- gress, 13: 95–109. malian CellsUsing Proteinase K and Phenol. Cold Spring White T.J., Bruns L., Lee S., Taylor J. (1990): Amplification Harbor, New York: 121–141. and direct sequencing of fungal ribosomal RNA genes for Sebastiana M., Pereira V.T., Alcântara A., Pais M.S., Silva A.B. phylogenetics. In: Innis M.A., Gelfaud D.H., Sninsky J.J., (2013): Ectomycorrhizal inoculation with Pisolithus tincto- White T.J. (eds): PCR protocols. A guide to methods and rius increases the performance of Quercus suber L. (cork applications. San Diego, Academic Press: 315–322. oak) nursery and field seedlings. New Forests, 44: 937–949. Wu Q.S. (2017): Arbuscular Mycorrhizas and Stress Toler- Sagheb-Talebi, K., Pourhashemi M., Sajedi T. (2014): Forests ance of Plants. In: Nadeem S.M., Khan M.Y., Waqas M.R. of Iran: A Treasure from the Past, a Hope for the Future. (eds): Arbuscular Mycorrhizas: An Overview. Singapore, In: Irano-Turanian Region. New York, Springer: 67–107. Springer: 1–24. Simonini G. (1992): Boletus comptus sp. nov. Rivista di Mi- Wurzburger N., Brookshire E.J., McCormack M.L., Lankau cologia, 35: 195–208. R.A. (2017): Mycorrhizal fungi as drivers and modulators Simonini G. (1998): Qualche Specie Rara O Poco Conosciuta of terrestrial ecosystem processes. New Phytologist, 213: Della Famiglia Boletaceae. In: Fungi non delineati. Michi- 996–999. gan, Mykoflora: 12–45. (in Italian with an English preface) Zambonelli A. (1993): An enzyme-linked immunosorbent Smith S.E., Read D. (2008): Arbuscular Mycorrhizae. In: assay (ELISA) for the detection of Tuber albidum ectomy- Smith S.E., Read D. (eds): Mycorrhizal Symbiosis. London, corrhiza. Symbiosis, 15: 71–76. Academic Press: 800. Received: March 28, 2020 Accepted: June 1, 2020

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Green space trends in small towns of Kyiv region according to EOS Land Viewer – a case study

Vasyl Yukhnovskyi1*, Olha Zibtseva2

1Department of Forests Restoration and Meliorations, Forest Institute, National University of Life and Environmental Sciences of Ukraine, Kyiv 2Department of Landscape Architecture and Phytodesign, Forest Institute, National University of Life and Environmental Sciences of Ukraine, Kyiv *Corresponding author: [email protected]

Citation: Yukhnovskyi V., Zibtseva O. (2020): Green space trends in small towns of Kyiv region according to EOS Land Viewer – a case study. J. For. Sci., 66: 252–263.

Abstract: The state of ecological balance of cities is determined by the analysis of the qualitative composition of green space. The lack of green space inventory in small towns in the Kyiv region has prompted the use of express analysis provided by the EOS Land Viewer platform, which allows obtaining an instantaneous distribution of the urban and suburban territories by a number of vegetative indices and in recent years – by scene classification. The purpose of the study is to determine the current state and dynamics of the ratio of vegetation and built-up cover of the territories of small towns in Kyiv region with establishing the rating of towns by eco-balance of territories. The distribution of the territory of small towns by the most common vegetation index NDVI, as well as by S AVI, which is more suitable for areas with vegetation coverage of less than 30%, has been monitored. We found that the share of dense vegetation in the territory of towns increased on average from 2.4 to 49.3% during 1990–2018. The share of the vegetation cover of moderate density decreased from 40.8 to 27.1%, and of sparse one from 37.5 to 14.9%. High variability of these indicators is noted. The share of open area for small towns decreased on average from 15.4 to 3.8%. The vegetation-free areas in 1990, 2005 and 2018 accounted for 3.8, 2.6 and 4.4%, respectively, which may indicate the intensive expansion of built-up areas over the last fifteen years. The development of urban greening systems was completely individual and depended not only on natural conditions but also on the manifestations of anthropogenic activity. The reduction of the ecological balance of the territories of small towns as of 2018 took place in the following sequence – Irpin, Tarashcha, Boiarka, Rzhyshchiv, Kaharlyk, Skvyra, Myronivka, Yahotyn, Uzyn.

Keywords: ecological balance; express analysis; rating; vegetation indices

The urbanized territory is a dynamic complex urban planning. First of all, the assessment of the that is constantly expanding and in need of ecologi- ecological balance of urban areas requires objective cal balance, making urban landscapes the most im- information on the types of land use and eco-stable portant area of environmental research. Long-dis- (green) areas. Unfortunately, researchers and plan- tance comparisons of landscaping systems require ners pay less attention to the study of small towns. detailed information on their relative abundance, Currently, not more than half of the small towns of vegetation composition and spatial structure (Ko- the Kyiv region have relevant master plans. Green- pecká et al. 2017). Understanding the patterns of ing schemes are not developed for them, and the urban landscaping and its spatial and temporal dy- inventory of green areas has never been conducted namics provides valuable information for general here due to the limited funds of local budgets. At

252 Journal of Forest Science, 66, 2020 (6): 252–263 Original Paper https://doi.org/10.17221/142/2019-JFS the same time, Ovcharenko and Zalyubovskaya (Small, 2001; Powell et al. 2007), as well as a rigid (2018) believed that traditional methods of land- classifier of urban vegetation by which it is identi- scape research are too cumbersome and costly, fied only as present or absent (Myint 2006). Usu- and the means of conducting them are outdated. ally, comparisons are used in studies (Foody, Boyd Instead, an up-to-date and promising area of green 1999). In doing so, Small (2001) did not prefer the space exploration is the use of open-source space normalized difference vegetation index (NDVI), imagery (Landsat 8, Sentinel-2) with a spatial reso- which is commonly used for global monitoring, but lution of 10–60 m, providing spatially consistent the estimation of vegetation shares. The integration coverage of large territories with high spatial detail of several methods on a medium-resolution scan- and periodicity. Based on such data, Rudenko et ner satellite image database allows accurate model- al. (2019) identified trends in land use in Ukraine’s ling of the vegetation fraction, despite the complex forest-steppe zone during the years 1992–2018, in mosaic of different types of urban cover (Gan et al. particular, the increase in the share of land under 2014). Data on the temporal dynamics of vegeta- construction, and revealed that the period of major tion are useful for in-depth studies of long-term changes occurred in 1994–2004. changes in urban greening, regional comparison The availability of satellite remote sensing data of cities with different economic and natural con- has increased significantly in recent decades, and is ditions, studying the relationship between urban now a powerful source for analysing the temporal greening and urbanization, obtaining information dynamics of urban conditions and quantifying ur- on sustainable urban development. ban environmental friendliness in a cost-effective The methodological framework used to study way (Deng et al. 2009; Liu, Yang 2013). At the same temporal and spatial changes in land use and land time, the increasing spatial resolution of commer- cover to meet the need for scientific information cial satellite imagery has influenced the emergence on sustainable development is evolving rapidly of new research and applications for urban applica- (Liu, Deng 2010). Specific features of the urban tions (Deng et al. 2009; Patino, Duque 2013). The landscape require further consideration of the pos- most common satellite applications include an sibilities and limitations of using satellite data and index of quality of life assessment, urban growth appropriate methods of analysis (Herold et al. 2005; analysis, assessment of the level of development, Patino, Duque 2013). urban social vulnerability and more. Li and Wenliang (2016) identified probable er- Patino and Duque (2013) established a correspon- rors in determining the impervious surfaces of ur- dence between vegetation indices for satellite data ban areas, in particular due to obstacles with large and urban green space inventory materials. The tree crowns. The use of satellite data in urban gov- possibilities of combining remote sensing data and ernance and, in particular, urban development, is a spatial metrics to analyse and model urban growth promising and effective method that is widely used and land use dynamics were investigated by Herold and opens new opportunities (Panarin, Panarin et al. (2005). The systematic combined use of these 2009). Remote sensing ground cover indices are the tools allows for a new level of information that im- most important urban indicators that contribute to proves understanding of urban dynamics and helps sustainable urban planning and management. develop alternative concepts of urban spatial struc- Considering that small towns are the most wide- ture development. According to Powell et al. (2007) spread and the least researched category of Ukrai- to monitor the evolution of the urban environment nian cities, where the inventory of green spaces has also requires the use of medium resolution multi- not been conducted at all, we consider the determi- spectral imaging. Such images are considered the nation of their territories as an example of a metro- best source of research because they provide the politan region extremely relevant. only unique long-term consistent set of digital data The purpose of the work is to determine the cur- (He et al. 2013). However, calculating classification rent state and dynamics of the ratio of vegetation and vegetation indices using medium spatial reso- and built-up area of small towns of the Kyiv region lution images may not be effective in quantifying and on their basis to establish the rating of towns physical information on urban landscaping. The by eco-balance of territories, which should extend fundamental problem of obtaining accurate satel- the researches we conducted earlier (Yukhnovskyi, lite data is the variability of the urban environment Zibtseva 2018; 2019).

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MATERIAL AND METHODS and the subsequent analysis and interpretation of the received data. Overall step-by-step description The study was based on the EOS Land Viewer, real- of this process supplemented by relevant screen- time image processing and analysis software that al- shots (Figures 1–4) is outlined below: lows obtaining the instant territory distribution across Step one of interaction with the software starts a range of vegetation indices and scene classification. by searching for a specific town. The software auto- We have considered the NDVI Territory Allocation to matically outlines the territory of the desired town. be the most commonly used, including for estimating In the second step (Figure 1), we search for a sat- urban areas. Also a modification of the soil-adjusted ellite image taken within the time of interest (year, NDVI‒S AVI index is used, which is recommended for month, and day). less than 30% vegetation area. The software used pro- At the next step (Figure 2) we select a combina- vides an instant division of the requested urban area tion of channels which are used to obtain various into a rectangle to which part of the suburban area surface characteristics (vegetation indices, types of also falls. coverage, etc.) The Land View service was used to obtain satel- Next, we obtain data on distribution of the ter- lite images (average resolution) of the studied small ritory of the studied town by types of vegetation, towns and surrounding areas at specified time peri- presence of open ground and absence of vegetation ods (year and decade of the month), capture scenes in percentage or absolute units of area (m2 or ha). and processed values of NDVI and S AVI indices. Examples of obtained data for the types of cover- Processing of NDVI and S AVI indices was carried age of Vyshneve town with percentage distribu- out by an automated software tool the types of cov- tion tables by NDVI and S AVI indices are shown erage with the transfer of the indicators in Excel in Figures 3 and 4, respectively.

Figure 1. Search for a satellite image

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ally shrubs and meadows), 0.1 – open ground, < 0.1 – vegetation is absent.

RESULTS AND DISCUSSION

Publicly available Google Earth satellite imagery (Figure 5) shows that suburban green zones (ideally in the form of a green ring) are not present around any small town. Usually, they are surrounded by ar- able land. Forests are only near Boguslav (mainly from the north), Tarashcha (from the northwest and southeast), Berezan (in the southwest), south of Pereyaslav, in the east of Tetiiv, northwest of Ukrainka, southwest of Fastiv and Boiarka, in the east west of Irpin, southwest of Vyshhorod, that is near half of the small towns in the region. The largest forests are near the town of Irpin. We tracked the dynamics of the percentage distribution of the territory of small towns of Kyiv region, obtained by NDVI and interpreted by different types of coatings, including different vegetation density as of 1990, 2005 and 2018. Figure 6 shows data for 1990 and 2018, which means that in 1990 there was sparse and moderate vegetation in the territory of towns. Quantitative values for towns varied greatly and did not allow high accuracy, but the average area of moderate vegetation was 40.8 ± 3.36%, sparse – 37.5 ± 2.15%, and dense (analogous to woodland) only 2.4 ± 0.74%. At the same time, the surface of the open ground averaged 15.4 ± 2.00%, and the Figure 2. Selection of a combination of channels area without vegetation (usually the surface of reservoirs) – 3.8 ± 1.86%. Such a wide range of fluctua- tions is caused by the presence or absence of reser- The above-described data collection process has voirs in urban areas, as rivers flow in the territories been carried out for the rest 19 small towns within of more than half of the small towns of Kyiv region. the same time frame or respective retrospectives. The average area of moderate vegetation in 2005 The main task was the simultaneous coverage increased by 8% and averaged 48.9 ± 2.31%, the and relative assessment of the territories of all area of sparse vegetation decreased by 12% and small towns of Kyiv region. The available Landsat amounted to 25.3 ± 1.83%, and the area of dense 8 or Sentinel 2 satellite data is usually evaluated vegetation increased by 14% – up to 16.7 ± 2.00%. from 26 to 28 August, given that early autumn is The average area of open ground decreased to 5.9 ± the best time for green space inventory. In addi- 1.32%, and without vegetation to 2.6 ± 1.43%. tion, the dynamics of the territories of twenty small The dense vegetation in 2018 was prevalent in towns was analysed according to satellite images in urban areas, the area of which increased by more the summer of 1995, 2005 and 2018. than 32% and averaged 49.3 ± 3.10% (V = 28.11; The EOS Land Viewer Web Interface forNDVI P = 6.12). The area of moderate vegetation de- provides the following automatic distribution creased by more than 21% compared to 2005 of the territory: 0.6–1 dense vegetation (trees), and amounted to 27.1 ± 1.66%, while the area of 0.4–0.5 moderate, 0.2–0.3 sparse (variable usu- sparse vegetation decreased by more than 10% to

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Figure 3. Distribution of the territory of Vyshneve by NDVI index

Figure 4. Distribution of the territory of Vyshneve by S AVI index

14.9 ± 1.71%. In this case, the area of ground cover presence of areas with sparse vegetation with cov- was 3.8 ± 0.77%, and without vegetation it varied erage below 30% is likely. That is why we have ap- most strongly, but less than in 2005 and increased plied the distribution of urban areas and S AVI. Fig- 1.7 times on average to 4.4 ± 1.90%. ure 7 shows the breakdown of the so-called “scene We assume that the dynamics of ground areas classification” that the EOS Land Viewer has been and areas without vegetation has also been affected providing since 2018, where towns are located in by the disadvantages of using the NDVI vegetation the order of the decreasing percentage of vegeta- indicator itself in urban environments, where the tion area.

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(A) (B)

(E) (F)

Figure 5. Nearly treeless areas of the towns of Yahotyn (A), Kaharlyk (B), Skvyra (C), Uzyn (D), Myronivka (E), Vyshneve (F) according to Google Earth Pro (Google, Mountain View, USA)

257 Original Paper Journal of Forest Science, 66, 2020 (6): 252–263

https://doi.org/10.17221/142/2019-JFS dense moderate sparse ground no vegetation 100 Figure 6. Dynam- (A) 90 ics of distribution of the territory 80 of small towns of Kyiv region by 70 types of coverage

) as of 1990 (A) and 60 %

( 2018 (B) 50 Area 40

0 dense moderate sparse ground no vegetation e v a v v v v v e k a v v v v v v k n n n n n n n dense moderate sparse ground no vegetation i i v ka ka v ka ka ii v h ha a ii v h ha y a t t l a l a l a l a k i k i h i

yra 2018 h i yra rka t rka

100 t s v s s ' v s n n s s rl y s s rl y i i rp i i rp i kh i i kh i a otyn a o t y l a orod h c orod h c a I i u I u y l Uz y Uz y r e y a r e h c h c s g s F s T e h F hn e y a T e hn e y a g s g Bu c g Bu c h a Sk v a Sk v (B) b u b u a h g a a h ro n ro n B o r a r a B e r e y s B e r e y s B o

90 Y a V Y a O K O V B o B o K e y s Ukr a Ukr a y s V V Rzy s Rzy s P e T a P T a M y V M y V 80 Small towns of Kyiv region Small towns of Kyiv region 70 ) )

% 60 % ( ( 50 Area Area 40

10 dense moderate sparse ground no vegetation

0 e v a v v v v v e k a v v v v v v k n n n n n n n i i e v a v v v v v e k a v v v v v v k n n n n n n v n ka ka v ka ka ii v h ha a ii v h ha i y a t i t l a l a l a l a k i v ka k i ka h i v

yra 2018 ka ka ii v h i h ha a yra t rka ii v h ha y rka a t t s v t s s ' v s l a l a n l a l a n k i s s k i h i rl y s s yra 2018 h i rl y yra t rka i rka t s i rp i v s s i ' rp i kh i v s i kh i n a n otyn s s a o t rl y s s y l a rl y orod h c i orod h c i rp i a I i i rp i kh i u I i kh i u a y l otyn Uz y a Uz y o t r e y a y l a r e h c orod h c h c orod s g h c a s I i u F s I T e h u F hn e y a y l T e Uz y hn e y a g Uz y r e s y a g r e Bu c h c g Bu c h a h c s g s F s Sk v T e h a F hn e y a Sk v T e hn e y a b u g s b u g Bu c g Bu c h a a h g a Sk v a a h Sk v ro n b u ro n b u B o r a r a B e r e a y s h g a B e r e y s B o a h Y a ro n V ro n Y a B o r a r a B e r e O y s B e K r e O y s B o V B o Y a B o K V e y s Ukr a Y a Ukr a y s O K O V V B o V B o K e y s Ukr a Rzy s Ukr a Rzy s y s P e V T a P V T a Rzy s M y V Rzy s M y V P e T a P T a M y V M y Small towns of Kyiv V region Small Stomwanlls tofw Knsy iovf rKegyiionv region Small towns of Kyiv region ) % ( At the beginning of the rating there are towns to the third, Vyshgorod – from the thirteenth to

Area such as Irpin, Tarashcha, Boiarka, Obukhiv, the seventh, Ukrainka – from the fi fteenth to the Vasyl’kiv, which are surrounded by suburban for- thirteenth, Yahotyn – from the twentieth to the ests, and at the end of the rating – Uzyn, Myroniv- eighteenth. Uzyn and Myronivka are the last in the ka, Kaharlyk, Skvyra – towns among the ploughed rating, also at the end of the rating are Kaharlyk, areas. Th e towns of Yahotyn, Ukrainka, Vyshhorod, Skvyra, and Vyshneve. Rzhyshchiv should be promoted in the eco-balance Figure 9 shows the dynamics of the vegetation rating signifi cantly ahead, given the large areas of cover of the territories of the explored towns for eco-stabilizing water surfaces, which also belong 1985, 1990, 2005 and 2018 and the ratings of the e v a v v v v v e k a v v v v v v k n n n n n n n i i v ka ka v ka ka ii v h ha a ii v h ha y a t t l a l a l a l a k i k i to green infrastructure. Such an adjusted rating is towns by the percentageh i of vegetation cover as of

yra 2018 h i yra rka t rka t s v s s ' v s n n s s rl y s s rl y i i rp i i rp i kh i i kh i a otyn a o t y l a orod h c orod h c a I i u I u y l Uz y Uz y r e y a r e h c h c s g s F s T e h F hn e y a T e hn e y a g s g shown in Figure 8. Bu c 1985 and 2018. g Bu c h a Sk v a Sk v b u b u a h g a a h ro n ro n B o r a r a B e r e y s B e r e y s B o Y a V Y a O K O V B o B o K e y s Ukr a Ukr a

y s Taking into account the water surfaces of Vyshneve now identifi ed among the towns with V V Rzy s Rzy s P e T a P T a M y V M y V Rzhyshchiv, it moved from the eleventh position the worst eco-balance, ranked top in 1985, indicat- Small towns of Kyiv region Small towns of Kyiv region 258 Journal of Forest Science, 66, 2020 (6): 252–263 Original Paper

https://doi.org/10.17221/142/2019-JFS

100100 Figure 7. Scenario of 100 coverage distribution vegetation non-vegetation water 90 vegetation non-vegetation water of the territory of small 80 80 80 towns of Kyiv region according to the 2018 607060 S AVI vegetation index 60 ) ) 40 40 % %

( 50 ( 204020 Area Area 30 0 200 10 0 Irpin Uzyn Fastiv Tetiiv Bucha Irpin Skvyra Uzyn Fastiv Tetiiv

Boiarka Fig. 7 Bucha Beresan Skvyra Yahotyn Obukhiv Vasyl'kiv Boguslav Kaharlyk Ukrainka Boiarka Fig. 7 Beresan Vyshneve Rzyshchiv Yahotyn Pereyaslav Obukhiv Tarashcha Vasyl'kiv Boguslav Kaharlyk Myronivka Ukrainka Vyshhorod Vyshneve Rzyshchiv Pereyaslav Tarashcha Myronivka Vyshhorod Small towns of Kyiv region Small towns of Kyiv region не рослинність 90 Small towns of Kyiv region Figure 8. Eco-balancing 80 rating of small towns by

) the percentage of green

% 70 ( infrastructure for 2018 60 50 40 30

Green Green space area 20 10 0 Irpin Uzyn Fastiv Tetiiv Bucha Skvyra Beresan Boyarka Yagotyn Vasylkiv Obukhiv Kagarlyk Boguslav Ukrainka Vyshneve Rzyshchiv Pereyaslav Tarashcha Vyshgorod Myronivka Small towns of Kyiv region

ing that the area’s ill-considered destructiveness centage of areas covered by dense and temperate over the course of the thirty years was indicative. vegetation for the eco-balance of urban areas. We Th e same applies to the territories of the towns of did not take any sparse vegetation into account due Skvyra and Uzyn. Instead, in 1985–2018, accord- to the signifi cant errors of this indicator, explained ing to the satellite distribution, the eco-balance of above. Vyshneve also leads the ranking in this di- the territories of the towns of Ukrainka, Rzhysh- vision. Tarashcha, Skvyra, Fastiv, Tetiiv are among chiv, Vyshhorod, Bucha, Tarashcha, Boiarka, and the leaders. Uzyn, Boguslav, Obukhiv, Vasyl’kiv, Irpin was improved. Th is was due to the increase Berezan are also among the best half of the list. in the density of plantations (growth of tree plants) Ukrainka, Rzhyshchiv, Vyshhorod, Bucha, Yahotyn over time in the towns and their surrounding ar- are closing the rating of the towns. eas and the smaller onset of development on the According to all the comparisons made by us woodlands. of the percentage distribution of the territories of Similar results are provided by the NDVI 1985 small towns, the towns of Irpin, Tarashcha, Boiar- territory analysis of towns, which sums up the per- ka, Rzhyshchiv top the list for 2018, and Kaharlyk,

259 Original Paper Journal of Forest Science,90 66, 2020 (6): 252–263

https://doi.org/10.17221/142/2019-JFS 2018 2005 1990 1985 80 90 70 2018 2005 1990 1985 80 60 70 ) 50 % ( рейтинг за рослинністю на 2018 рік 60 Area 40 ) 50 % 30 ( рейтинг за рослинністю на 2018 рік Area 40 20 30 10 20 0 Irpin Uzyn 10 Fastiv Tetiiv Bucha Skvyra Beresan Boyarka Yagotyn Vasylkiv Obukhiv Kagarlyk Boguslav Ukrainka Vyshneve Rzyshchiv Myrinivka Pereyaslav Tarashcha Vyshgorod 0 Irpin Uzyn Fastiv Tetiiv Bucha Skvyra Beresan Boyarka Yagotyn Vasylkiv Obukhiv Kagarlyk Boguslav Ukrainka Vyshneve Rzyshchiv Myrinivka Pereyaslav Tarashcha Vyshgorod 90 рейтинг за рослинністю на 1985 рік 2018 2005 1990 1985 (A) (B) 80 90 рейтинг за рослинністю на 1985 рік 70 2018 2005 1990 1985 80 60 70 50 ) % ( 60 Area 40 50 ) % 30 ( Area 40 20 Fig 9 30 10 20 Fig 9 0 Irpin Uzyn 10 Fastiv Tetiiv Bucha Skvyra Berezan Boyarka Yagotyn Vasylkiv Obukhiv Kagarlyk Boguslav Ukrainka Vyshneve Rzyshchiv Figure 9. Rating of towns by percentage of land cover for 1985 (A) and 2018 (B) for 9. Rating of townsland by percentage cover Figure Pereyaslav Tarashcha Vyshgorod Myronivka 0 260 Irpin Uzyn Fastiv Tetiiv Bucha Skvyra Berezan Boyarka Yagotyn Vasylkiv Obukhiv Kagarlyk Boguslav Ukrainka Vyshneve Rzyshchiv Pereyaslav Tarashcha Vyshgorod Myronivka Journal of Forest Science, 66, 2020 (6): 252–263 Original Paper https://doi.org/10.17221/142/2019-JFS

Dendrogram for 20 observations Figure 10. Dendrogram of similarity of Full communication method small towns of Kyiv region according Eucliden distance to the classification of scenes for 2018 6

2 Distance combined 1

0 Irpin Uzyn Fastiv Fastiv Tetiev Bucha Bucha Skvyra Boiarka Berezan Yahotyn Obukhiv Vasyl’kiv Vasyl’kiv Kaharlyk Kaharlyk Boguslav Boguslav Ukrainka Vyshneve Rzyshchiv Tarashcha Tarashcha Pereyaslav Myronivka Vyshhorod

Small towns of Kyiv region

Skvyra, Myronivka, Yahotyn, Uzyn are closing the moved from the sixteenth to the twelfth position list. It is in these small towns that the actual photos (and for all green infrastructures – to the seventh). of the territories as treeless are shown in Figure 5. Cluster analysis of urban areas dividing the clas- The situation with green infrastructure, and there- sification of scenes into water surfaces, vegetation fore with eco-balance, is the worst. and “non-vegetation” (other types of cover) as of Vyshneve and Uzyn were among the best in eco- 2018 allowed to obtain a dendrogram (Figure 10). logical balance (percentage of vegetation coverage) According to the obtained data the studied small for 1985. They have now significantly lost their towns may be subdivided into two groups. The first positions and are now at the end of the ranking. group consists of six towns which are characterized Bucha and Ukrainka, Rzhyshchiv, Vyshhorod, the by relatively better indicators: a larger area of vege- towns along the Dnieper River with significant in- tation and water surfaces (i.e. more developed green clusion of the surface of reservoirs in the territorial infrastructure); these are the towns of Ukrainka, boundaries, are among the worst cities in terms of Vyshhorod, Rzhyshchiv, Boiarka, Tarashcha and ecological balance. Irpin. The remaining fourteen towns belong to the According to the distribution by soil-adjusted second group. Figure 11 shows the clusters of stud- S AVI, the town of Vyshneve moved in the time dy- ied towns according to the classification of scenes. namics from the first position in 1985 to the fifth Cluster analysis also identified four smaller sub- in 1990, ninth in 2005, fourteenth in 2018 and tak- groups (clusters). The first cluster included three ing into account all green infrastructures – to the towns: Rzhyshchiv, Vyshhorod and Ukrainka. They fifteenth position. The town of Skvyra moved from are located directly on the right bank of the Dnieper second to fourteenth position. River and have large areas of water surfaces and The town of Uzyn moved from third to second vegetation cover. The second cluster includes eight position, then thirteenth and nineteenth (and tak- towns with the smallest share of water surfaces, but ing into account all green infrastructures – to the with the largest share of vegetation cover. These are last twentieth position). Instead, Ukrainka gradu- the towns of Irpin, Tarashcha, Boiarka, Obukhiv, ally rose from the last position to the fifteenth Vasyl’kiv, Fastiv, Bucha, Berezan. These towns (and for all green infrastructures – to the twelfth). are surrounded by large forests. The third cluster Rzhyshchiv moved from the penultimate to the six- includes six towns: Pereyaslav, Boguslav, Tetiiv, teenth, fourteenth, and tenth position (for all green Vyshneve, Skvyra, Kaharlyk, with average values infrastructures – the third level) and Vyshhorod of vegetation cover. The fourth cluster consists of

261 Original Paper Journal of Forest Science, 66, 2020 (6): 252–263

https://doi.org/10.17221/142/2019-JFS

Figure 11. Clusters of studied towns according to the classification of scenes (1 – Rzhy- shchiv, Vyshhorod, Ukrainka; 2 – Irpin, Tarashcha, Boiarka, Obukhiv, Vasyl’kiv, Fastiv, Bu- cha, Berezan; 3 – Pereyaslav, Boguslav, Tetiiv, Vyshneve, Skvyra, Kaharlyk; 4 – My- ronivka, Uzyn, Yahotyn)

three small towns: Myronivka, Uzyn, and Yahotyn dense plantations from 2.4 to 49.3%. At the same with a minimum share of vegetation. time, the share of ground cover in the experimental It is advisable to create forests in their suburban urban territories decreased from 15.4 to 3.8%, and areas to increase the eco-balance of small towns. the areas without vegetation increased on average from 3.8 to 4.4%. CONCLUSION The development of green plantations in small towns was completely individual and depended not It is justified that in the context of current data only on natural conditions but also on the directions constraints on small-scale green space systems of anthropogenic activity. As of 2018, the rating of the the medium resolution images obtained from the eco-balance of urban areas of Irpin, Tarashcha, Boiar- Landsat 8 and Sentinel 2 satellites are informative ka, Rzhyshchiv is at the top, and Kaharlyk, Skvyra, enough. Snapshots of different time periods allow Myronivka, Yahotyn, Uzyn are closing the list. you to track the simultaneous dynamics of green In order to increase the level of ecological bal- coverage, and hence the eco-balance of small cities ance of urban areas, one of the most urgent mea- and to perform their comparative analysis. sures is to create broad protective and recreational Extremely convenient for landmark large-scale strips in the form of suburban forests, which is comparative studies is the EOS Land Viewer soft- not problematic in the studied Polissya and forest- ware with the ability to instantly split the study steppe conditions. areas by vegetative and scene classification. It was found that the most correct for the overall assess- ACKNOWLEDGMENT ment of the ecological balance of urban areas were S AVI indicators with a percentage of green infra- We express our sincere gratitude to the EOS Cal- structure, as well as NDVI with a percentage of ifornia Company (https://eos.com/landviewer) for dense and temperate vegetation. the free access to the images and use of the Land Studies have shown that in the territory of small Viewer service and to the Head of the Rivne Re- towns of Kyiv region for the period from 1985 to gional State Administration – alumnus of the For- 2018, the percentage of areas under sparse stands estry Faculty of our University to Oleksandr Danyl- was from 37.5 to 14.9% and it increased under chuk for support of the research.

262 Journal of Forest Science, 66, 2020 (6): 252–263 Original Paper https://doi.org/10.17221/142/2019-JFS

REFERENCES Ovcharenko A., Zalyubovskaya O. (2018): Indicative landscape monitoring of national natural parks (on the example Deng J., Wang K., Hong Y., Qi J. (2009): Spatio-temporal of the territory of Slobozhansky National Nature Park). dynamics and evolution of land use change and landscape Bulletin of Kharkiv National University, series “Geology, pattern in response to rapid urbanization. Landscape and Geography, Ecology”, 49: 190–205. (in Ukrainian) Urban Planning, 92: 187–198. Panarin V., Panarin R. (2009): The use of space images in Foody G., Boyd D. (1999): Detection of partial land cover the municipal management of urban areas for territorial change associated with the migration of inter-class tran- planning purposes. Geomatics, 3, 40–55. (in Ukrainian). sitional zones. International Journal of Remote Sensing, Patino J., Duque J. (2013) A review of regional science ap- 20: 2723–2740. plications of satellite remote sensing in urban settings. Gan M., Deng J., Zheng X., Hong Y., Wang K. (2014): Moni- Computers, Environment and Urban Systems, 37: 1–17. toring urban greenness dynamics using multiple endmem- Powell R., Roberts D., Dennison P., Hess L. (2007): Sub-pixel ber spectral mixture analysis. PLoS ONE, 9: e112202. mapping of urban land cover using multiple endmember He C., Convertino M., Feng Z., Zhang S. (2013): Using LiDAR spectral mixture analysis: Manaus, Brazil. Remote Sensing data to measure the 3D green biomass of beijing urban of Environment, 106: 253–267. forest in China. PLoS ONE, 8: e75920. Rudenko L., Golubtsov O., Chekhniy V., Tymuliak L., Farion Herold M., Couclelis H., Clarke K. (2005): The role of spatial Yu. (2019): Landuse changes in the forest-steppe zone of metrics in the analysis and modeling of urban land use Ukraine during 1991–2018: methodology of research and change. Computers, Environment and Urban Systems, main trends. Ukrainian Geography Journal, 1: 24–32. 29: 369–399. Small C. (2001): Estimation of urban vegetation abundance by Kopecká M., Szatmári D., Rosina K. (2017): Analysis of ur- spectral mixture analysis. International Journal of Remote ban green spaces based on sentinel-2A: case studies from Sensing, 22: 1305–1334. Slovakia. Land, 6: 25. Wenliang L. (2016): “Large-Scale Urban Impervous Surfaces Liu J., Deng X. (2010): Progress of the research methodolo- Estimation Through Incorporating Temporal and Spatial gies on the temporal and spatial process of LUCC. Chinese Information into Spectral Mixture Analysis” Theses and Science Bulletin, 55: 354–362. Dissertations: 110. Liu T., Yang X. (2013): Mapping vegetation in an urban area Yukhnovskyi V., Zibtseva O. (2018): Dynamics of ecological with stratified classification and multiple endmember stability of small towns in Kyiv region. Journal of Geology, spectral mixture analysis. Remote Sensing of Environment, Geography and Geoecology, 27: 2. 133: 251–264. Yukhnovskyi V., Zibtseva O. (2019). Estimation of ecological Myint S. (2006): Urban vegetation mapping using sub-pixel stability of small town Bucha in Kyiv region. Ukrainian analysis and expert system rules: A critical approach. In- Geography Journal, 2: 49–56. ternational Journal of Remote Sensing, 27: 2645–2665. Received: December 5, 2019 Accepted: June 19, 2020

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