Assessment of Forest Pests and Diseases in Protected Areas of Final report

Dr. Iryna Matsiakh Tbilisi 2014

This publication has been produced with the assistance of the European Union. The content, findings, interpretations, and conclusions of this publication are the sole responsibility of the FLEG II (ENPI East) Programme Team (www.enpi-fleg.org) and can in no way be taken to reflect the views of the European Union. The views expressed do not necessarily reflect those of the Implementing Organizations.

CONTENTS

LIST OF TABLES AND FIGURES ...... 3 ABBREVIATIONS AND ACRONYMS ...... 6 EXECUTIVE SUMMARY ...... 7 Background information ...... 7 Literature review ...... 7 Methodology ...... 8 Results and Discussion ...... 8 and Kintrishi Protected Areas ...... 8 ...... 9 Recommendations ...... 9 Other findings and Research ...... 10 INTRODUCTION ...... 12 1. BACKGROUND INFORMATION ...... 13 1.1. Status of Biodiversity ...... 13 1.2. Protected Areas ...... 15 1.3. Brief description of the research areas ...... 17 2. LITERAT RE REVIEW ...... 22 2.1. A brief description of the physical geographical and the sanitary conditions of U colchica in Мtirala National Park and Kintrishi Protected Areas ...... 22 2.2. Boxwood Diseases ...... 23 2.2.1. Shoot Blight of Boxwood – Calonectria pseudonaviculata (anamorph Cylindrocladium buxicola) ...... 23 2.2.2 Other Boxwood Diseases ...... 27 2.3. Boxwood Pests ...... 30 2. 2. A brief description of the physical‐geographical and the sanitary conditions of Imeretian in Ajameti managed reserve ...... 31 2.4. Imeretian Oak Diseases and Pets ...... 33 3. Methodology ...... 36 3. 1. Field Research ...... 36 3. 2. Laboratory Work ...... 37 4. RESULTS AND DISCUSSION ...... 40 4.1 Mtirala National Park ...... 40 4.2. Kintrishi Protected Areas ...... 45 2

4.3. Ajameti Managed Reserve ...... 49 5. Recommendations ...... 66 5.1. Proposals for the conservation management on the territory of Kintrishi Protected Areas and Mtirala National Park ...... 66 5. 2. Proposals for the conservation management on the territory of the Ajameti managed reserve ...... 68 5.2.1. Protection and conservation the best‐preserved areas of old‐growth Imeretian oak stand ...... 68 5.2.2. Restoration of forests with a predominance of Imeretian oak ...... 70 5.2.3. Environmental, educational and recreational activities ...... 71 Other Findings and Research ...... 73 References ...... 81 Annex A...... 97 Mtirala National Park ...... 97 Annex B ...... 100 Kintrishi Protected Area ...... 100 Annex C ...... 102 Ajameti Managed Reserve ...... 102

LIST OF TABLES AND FIGURES

Table 1. IUCN category status of some of the further research areas in Georgia ...... 16 Table 2. IUFRO classification ...... 36 Table 3. Factors affecting the deterioration of health conditions of oak forests in Ajameti ...... 53 Table 4. The list of found in Imeretian oak stands of Ajameti managed reserve ...... 55 Table 5. Pathogens composition found in Ajameti managed reserve ...... 57

Figure 1: Map of forest distribution in Georgia (data of National Environmental Action Programme of Georgia 2012 –2016) ...... 13 Figure 2: Map of the distribution of protected territories in Georgia (data of National Environmental Action Programme of Georgia 2012 –2016) ...... 15 Figure 3: Location of Мtirala National Park (data of Agency of Protected Areas) ...... 17 Figure 4: Location of Kintrishi Protected Areas ...... 18 Figure 5: Location of Ajameti managed reserve ...... 19 Figure 6: The dark or light circle spots on the boxwood leaves (by Ivors K.) ...... 26 Figure 7: Black cankers on the stem (by Ivors K.) ...... 26 Figure 8: Laboratory work ...... 38 Figure 9: The baiting technique for Phytophthoras isolation ...... 38 Figure 10:. Boxwood decline along the river in Mtirala National Park ...... 40 Figure 11:Boxwood decline in the natural boundary in Mtirala National Park ...... 40 Figure 12: Isolation results of baiting technique from boxwood forests in Colchida lowland ...... 41 3

Figure 13: symptoms on the rhododendron leaves: a – Phytophthoras damages on the leaves; b – brown discoloration of leaves tissues caused by Phytophthora and other saprotrophic ...... 42 Figure 14: Phytophthoras symptoms on the main stem of Buxus colchica a, b - black spots and blooding on the outside of the bark; c, d, - dark brownish-black coloration of the vascular tissues under the bark; e - black stripes in the wood; f - affected inner tissues of wood by Xyleborinus saxeseni Ratz...... 43 Figure 15: Reducing the growth of boxwood leaves and branches ...... 43 Figure 16: Fruit bodies of fungus: a – versicolor (L.) Lloyd, b– Stereum subtomentosum Pouzar...... 44 Figure 17: Calonectria pseudonaviculata on the boxwood leaves: a, b - the fungus Calonectria pseudonaviculata on the boxwood leaves; c - isolate of Calonectria pseudonaviculata on PDA media ...... 44 Figure 18: Volutella buxi on boxwood leaves: a, b – sporification on the leaves; c- isolates of Volutella buxi on PDA media ...... 45 Figure 19: Decline of Buxus colchica in Kintrishi Protected Areas ...... 46 Figure 20: Damaged boxwood tree stands in Buxus colchica grove ...... 46 Figure 21: The native forest regeneration on the main stems of boxwood ...... 46 Figure 22:The poor growth of Buxus colchica branches and twigs ...... 46 Figure 23: Formation of new vegetation succession under canopy of dead boxwood trees...... 47 Figure 24: The damage of boxwood leaves ...... 47 Figure 25: Eriococus buxi Fousc. on the boxwood leaves ...... 47 Figure 26: Damaged boxwood leaves by near Administartion of Mtirala National Park: a – injured , b – larva...... 49 Figure 27: Distribution of oak trees at heights classes ...... 50 Figure 28: Distribution of oak trees at classes of vitality ...... 50 Figure 29: Distribution of oak trees at hierarchical trend ...... 51 Figure 30: Distribution of oak trees at silvicultural value ...... 51 Figure 31: Distribution of oak trees at the quality of bole (trunk) ...... 52 Figure 32: Distribution of oak trees at the crown lenght ...... 52 Figure 33:The development of stem rot in coppice origin ...... 53 Figure 34: Imeretian oak leaves damage by late spring frost ...... 54 Figure 35: The number of trees with frost cracks and timbers ...... 54 Figure 36: Root rot in the frost cracks places ...... 54 Figure 37: Mines of leaf-mining on the surface of oak leaves ...... 56 Figure 38: Oak leaves damaged by caterpillars Dyseriocrania subpurpurella ...... 56 Figure 39: formed by Silk button ( numismalis) ...... 57 Figure 40: The pineal galls are formed by by Andricus foecundatrix ...... 57 Figure 41: Imeretian oak leaves affected by powdery mildew ...... 57 Figure 42: Sanitary conditions of the natural regeneration of oak self-seedlings: a - in June; b – in October ...... 59 Figure 43: Infected of oak self-seedling: a – in June; b – in October...... 59 Figure 44: The Phytophthora symptoms on the young Imeretian oak trees ...... 60 Figure 45: Isolation results of soil baiting technique from Imeretian oak forests in Colchida lowland ...... 60 Figure 46: Number of trees with top drying and dried main branches ...... 62 Figure 47: The distribution of trees by Category of the sanitary condition ...... 62 Figure 49: The remains of imago great capricorn ...... 63 Figure 48: Flying holes of great capricorn beetle ...... 63 Figure 50. Number of trees with signs of settling great carpicorn beetle ...... 63 Figure 51: Imago flying holes on the trees with different categories of the health status ...... 63 Figure 52: Rotten wood as a of saprotrophic organisms ...... 64 Figure 53: The lesser stag beetle Dorcus parallelipipedus L. – the typical inhabitant of rotten wood of hardwood tree spesies ...... 64 4

Figure 54: Shpyhovka (sowing) acorns under the blade (by Kolesnychenko, 1981) ...... 71 Figure 55: Phytophthora symptoms on the main stems ...... 73 Figure 56: Phytophthora symptoms on Carpinus orientalis on the main stems ...... 73 Figure 57: Phytophthora symptoms on Juglans spp. main stems...... 74 Figure 58: Decline of in Mtirala National Park ...... 74 Figure 59: Symptoms of Cryphonectria parasitica on the affected chestnut shoots ...... 75 Figure 60: Isolates of Cryphonectria parasitica obtained from affected chestnut shoots ...... 76 Figure 61: Phytophthora symptoms on the sweet chestnut leaves ...... 76 Figure 62: ekebladella (Bjerkander). on the leaves chestnut ...... 77 Figure 63: Lobaria pulmonaria in Mtirala National Park: a – the thallus of Lobaria pulmonaria, b - leathery and lobed with a pattern ridges and depressions on the upper surface, c - the thallus of Lobaria pulmonaria on the bark of Imeretian oak in Ajameti managed reserve ...... 77 Figure 64: Brown pycnidia on the surface of necrosis lesion ...... 79 Figure 65: Cancer lesion on the trunk of a poplar ...... 79 Figure 66: The larva of aspen green sawfly ...... 80 Figure 67: Rust on the surface of the poplar leaves ...... 80

About the Author Dr. Iryna Matsiakh is the Doctor of Biological Science from Lviv, . Mrs. Matsiakh holds a PhD degree in forest pathology and silviculture from the National Forestry University of Ukraine. With 10 years of experience, Mrs. Matsiakh works on field research and laboratory study of different types of forest pathologies. Mrs. Matsiakh is the author of many publication on this topic.

The cover picture and photos in the next report were taken by the executor of these research. 5

ABBREVIATIONS AND ACRONYMS

APA Agency of Protected Areas BLAST Basic Local Alignment Search Tool DNA Deoxyribonucleic acid EPPO European and Mediterranean Plant Protection Organization ENPI European Neighbourhood and Partnership Instrument EU European Union FLEG Forest Law Enforcement and Governance ha hectares ITS Internal transcribed spacer IUCN International Union for Conservation of Nature IUFRO International Union of Forest Research Organizations MAS 2% Malt-agar with streptomycin NP National Park SNR Strickt Nature Reserve PA Protected Areas PARPH Selective medium with antibiotics PARPNH Selective medium with hymexazol for Phytophthora species PCR Polymerase Chain Reaction PDA Potato-dextrose agar PDS Potato-dextrose agar with streptomycin spp. As a short way of saying that something applies to many species within a The WB The World Bank WWF World Wile Fund for Nature

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EXECUTIVE SUMMARY

This report is the final report of the researches conducted for the assessment of forest pests and diseases in Protected Areas of Georgia. The work was carried out from June to December 2014. Background information

Georgia’s protected areas deserve special attention because the total area has significantly increased during the recent years (National Environmental Action Programme of Georgia, 2012). 7.3% (511,122.5 hectares) of all of Georgia’s territory was protected by law before 2011. The Law on System of Protected Areas adopted in 1996 regulates all protected ecosystems in Georgia. Following the International Union for Conservation of Nature (IUCN) classification, the Law contains references to IUCN categories and has established a system of natural reserves, national parks, protected landscapes, biosphere reserves and others, and determines a regime for their use and protection (Introductory Report on Nature Conservation in Georgia, 2010; Biodiversity assessment for Georgia, 2000). In 1996, the protected areas categories that are in compliance with international, IUCN classification system have been accepted (Biodiversity assessment for Georgia, 2000). In addition, the Ministry of Environment and Natural Resources Protection of Georgia represents and shows internationally accepted categories of protected areas based on IUCN (Introductory Report on Nature Conservation in Georgia, 2010). Categories of PAs of Georgia include: • Strict Nature Reserve/State Reserve, IUCN Category I; • National Park, IUCN Category II; • Natural Monument, IUCN Category III; • Managed Reserve/Sanctuary, IUCN Category IV; • Protected Landscape, IUCN Category V; • Multiple-Use Territories, IUCN Category VI. Mtirala National Park and Kintrish Protected Areas were selected for the assessment of the sanitary conditions of the evergreen Tertiary period relic plant Buxus colchica (IUCN Red List of Threatened Species; Category CR; Criteria String: A1a; B1b (i)) and Ajameti managed reserve where the absolute dominant species is an ancient, rare and relict Imeretian Oak (Quercus imeretina) (IUCN Red List of Threatened Species) which occupies 95% of the area. According to the data collected in 2004, 192900 ha of Georgia’s forests suffer from various diseases and pathologies studies of the forests have not been conducted in the recent years with the exception of episodic studies in some protected areas. Literature review The literature review and analysis of the existing information about the boxwood decline and Imeretian oak dieback around the world and in Georgia were conducted. Geographic range of Buxus colchica is concentrated in the from North Kolkheti in Russia to South Kolkheti in . It was known that Buxus colchica is resistant to pests and diseases (http://kraevedenie.net/2009/03/19/samshit-buxus/2/) but the research observation conducted from 2009 shows another situation. The first signs of Buxus colchica damage were found in 2009. In 2008 there was a very dry summer and then rainy wet spring in 2009. It was proved that in 2010, there was the climate change and it was the driest year at the century but at the same time with a very low humidity. Because of these reasons Buxus colchica disease caused by Cylindrocladium buxicola was revealed (Gorgiladze et al. 2011; Matchutadze et al. 2013). Moreover, nowadays it is still in the great danger. Later in 2011 in Kintrishi Protected Areas and Mtirala National Park, the boxwood trees were examined and leaf loss and discoloration of stem was observed. The two areas of 0.6 ha were selected for the research in Mtirala National Park where 60% of Buxus colchica dieback was found. On the third research plot 18 trees were planted but in a short time 75% of them were infected. For the third research, 0.009 ha were selected in Kintrishi Protected Areas for the investigation of 7 boxwood trees diseases and 55-65% of damage was found (personal communication with Khatuna Tsiklauri, Agency of Protected Areas, Georgia). In 2011, all reports in Georgia informed about a new disease that was not previously determined - box blight caused by Cylindrocladium buxicula. Also, in the literature review the information about other possible pathogens and pests of Buxus spp. was studied and described in details. The process of oak forests dieback (decline) has been the focus of foresters for more than 100 years. The cases of oaks dieback in have been observed occasionally since 70s of the XIXth century (Rykowski, 1997). Researchers often agree with the idea that dieback oak effect is caused by complex factors, including the impact of extreme weather conditions, changes in the hydrological regime, damage of the oak phytophagy and disease, human impact etc. (Osipov et al. 1989; Tsaralunha, 2002, 2005; Kätzel et al. 2006; Selochnik, 2008). A certain role in the process of degradation of the oak forests belongs to the disease, which play a secondary role (Selochnik, 1998, 1999, 2002). Oak decline disease was first detected in Georgia in the 1970s and was associated with Verticillium sp., Rhinotrichum spp., Erwinia multivora and E. lignifilla on Quercus imeretina, Armillaria mellea on Q. iberica, and leaf mining insects (green oak roller moth (Tortrix viridana), winter moth (Operophtera brumata), oak umber (Erannis leucophaearia) were found on both Quercus spp. (Imnadze et al. 1989) During examination of the oak forest stands in Ajameti managed reserve in 2007 by the researches of Vasil Gulisashvili Forestry Institute 12 species of pathogenic fungi and 13 species of pests were found. Among them, were identifined cerdo acuminatus which damages trees and Ganoderma applanatum which penetrates through outlet openings of beetle adults and leads to stem (komlevoyi) rotten wood (Report on forest pathology, 2007). The biological and ecological features of great capricorn beetle and other pathogens of Imeretiam oak were studied in great details and represented in the literature review enough well. Methodology The methodology of study was divided into two parts: field research and laboratory work. The field research was conducted in June and October 2014 on the territories of examined Protected areas.The study of the sanitary condition of forest plantations was conducted using the forest-pathological examination (Vorontsov et al. 1991). IUFRO tree classification was used for the measuring tree characteristics (Leibundgut, 1958). The diseases and damages of tree stands were studied following recommendations of Rudnev (1957), Tsaralunha and Kaharmanova (2006), Furmenkova (2009), Sanitary rules… (1995). The processing and the preparation of the collected samples were carried out in the Laboratory of Forest Protection of Forestry University of Ukraine for further laboratory analysis. The soil and tissues samples of symptomatic Buxus colchica and Quercus imeretina plants were analyzed in (DIBAF University of Tuscia, Viterbo) and in (Forest Research institute, Warsaw) and different approaches were used. Phytophthoras were isolated from soil samples using the baiting technique (Jung et al. 1996). DNA extraction from soil was carried out using the Power Soil® DNA isolation kit (MoBio Laboratories Inc, CA, USA) according to the manufacturer’s protocols. Extraction of genomic DNA from was performed as reported by Lee and Taylor (1990). The PCR was carried out using the ITS1-ITS4 primers (White et al. 1990). Amplicons were sequenced (Macrogen, Amsterdam, The Netherlans)) and the Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/) was used for sequence similarity searches. Results and Discussion Mtirala National Park and Kintrishi Protected Areas The massive decline of boxwood forests was observed in Mtirala National Park and Kintrishi Protected Areas during the field trips. According to the results of the field and laboratory studies conducted in Mtirala National Park and Kintrishi Protected Areas in boxwood forests stands the strong development of soil phytopathogens of Phytophthora genus, which cause root rot, was observed. 220 isolates of Phytophthoras (from the soil of boxwood forests) were obtained using baiting technique. Identification of Phytophthora on these two objects is not random. The climatic conditions of the regions and sufficient moisture contribute to the spread of these pathogens. In 8

Mtirala National Park as well as in Kintrishi Protected Areas the examined boxwood stands grow on the banks of rivers and root systems of the plants are periodically flooded in spring and during the period of heavy rains. Calonectria pseudonaviculata was isolated directly from the sporulation on the leaves and mycelium on shoots. However, the detail information about Calonectria pseudonaviculata is not emphasized as this fungus had been already detected and its role in the decline of boxwood tree stands was described (Gorgiladze et. al. 2011). Volutella box blight - Volutella buxi (teleomorph Pseudonectria rouselliana) was found after keeping damaged boxwood leaves in a moist chamber that was also isolated in culture. During the second visit to Georgia, the damage of box tree by caterpillars of Cydalima perspectalis was identified in green areas near the office of Kintrishi Ptotected Area (Kobuleti) and administration of Mtirala National Park (village Chakvi), as well as in the city of and Tikeri nursery farm (Administration of Kobuleti, Lepl Forestry Agency of Ajara).

Ajameti managed reserve The results of the evaluation the general condition of Imeretian oak stands present the results of the distribution provisions in the forest communities. The majority of trees (55-65%) is related to the middle of the tier and their height is 1/3-2/3 of the maximum height of forest stands. In the examined stands in the middle and on the edge of forests the tree of good vitality are prevailed in Ajameti and and their number is 47-60%. The total number of trees of poor vitality is higher in stands located on the edge of forests. The trees not modifying the social position (47-68%) are dominated in old-aged Imeretian oak stands. There is a number of trees (8-35%) that may worsen their social position. In the surveyed plots, the helpful concomitant trees that contribute to the growth of elite trees are dominated and the number of elite trees ranges from 10 to 25%. There is a very significant development of stem rot that reduces the quality of the wood. The number of trees of good quality increases from 32.5% at the edge of the forest to 42.5% in the centre of the area in Ajameti research plot. A well-developed canopy of trees increases the resistance of trees to external influences. The examined Imeretian oak stands on the site of Ajameti and on the central areas of Vartsikhe are preferably formed of well-developed crown and old-aged trees. According to the Manoins’ theory, three groups of factors that can work in parallel and in series were distinguished and their impact on the Imeretian oak tree stands in Ajameti managed reserve was analyzed. The lists of -foliophagi and the species composition of pathogenic fungi affecting necrosis and rot pathogens found in Imeretian oak stands were given and described with pictures. 21 species of pathogenic fungi were discovered on the territory of Ajameti. Using baiting technique 163 isolates of Phytophthora from soil of Imeretian oak forests were obtained. Isolation of Phytophthora on the territory of Ajameti actually causes concern. About oak decline in Europe had been known for a long time, as well as about decline of Imeretian oak started to talk 100 years ago. This information about the possible causes of this phenomenon is described in details in the literature review. However, never Phytophthora was discovered and described in the Imeretian oak forests of . Also, the distribution of trees by Category sanitary condition was evaluated connected with the development of a great capricorn beetle. During research, any flying holes of a great capricorn beetle on alive trees with no signs of weakening were not found (I Category sanitary condition). With the deteriorating of trees, the health conditions the number of flying holes increases. Discussion about the role of dead wood in the forest on the basis of the following requirements of the WWF and old-aged oaks forests in Ajameti was analyzed and the reasons of protection and conservation these forests were described.

Recommendations The treatment measures and conducting some control of pathogens development in the natural boxwood tree stands is a very difficult task. Because of using fungicides on the territories of protected areas may have a negative impact on the environment (particularly on mycorrhizae, soil invertebrates and aquatic organisms). The protected status of research objects (Kintrishi protected 9

area - IUCN category I and Mtirala National Park - IUCN category II) also does not allow using actively fungicides. Also, it is concerned about penetration in the neighboring zones of protected areas the new invasive species butterfly Cydalima perspectalis. One way of treatment against caterpillars of Cydalima perspectalis in green areas is using the insecticide of enteric contact action, particularly synthetic pyrethroids: Sumi-alpha (Esphenvalerat, Senpai); Decis, Decis prophi (Kotryn, Oradelt, Polytox,) Fastak (Alphatoks, Geletrin, Kinimks, Fury) and others. In our opinion, on the territory of studied areas, the dieback of the existing boxwood trees has already reached an irreversible process stage. Despite this, in order to restore box tree stands the several tasks were recommended. In our opinion, the conservation activities on the territory of the Ajameti manage reserve should be developed according to the following three directions: 1. Protection and conservation of the best-preserved areas of old-growth Imeretian oak stands. 2. Restoration of forests stands with a predominance of Imeretian oak in trees composition. 3. Activities in the field of environmental education, tourism and recreation. The smaller clusters – such as Vertsikhe and Sviri - require a different forest management regime. The forest management must correspond to the state of Imeretian oak stands in different parts of the reserve in order to be able to decide whether the selection of certain areas and the possible mode of economic intervention are reasonable. On the territory of Ajameti managed reserve there is a conflict of interests between the two species of the Red List of IUCN, which should be protected, Imeretian oak and great capricorn beetle (Cerambyx cerdo). That is why we think that the active measures against great capricorn beetle in Ajameti are not needed. Greater attention should be focused on of Imeretian oak stands by promoting its natural regeneration or, if necessary, planting oak forests. For the derived plantings that had emerged because of forest management in previous decades, it is worth to provide reconstruction of these tree stands. The reconstructive measures of derived plantations should be conducted with a view to reproduce the native forest tree stands. For future conversation and restoration of Imeretian oak strict control and monitoring visits to Ajameti reserve should take place. Therefore, there is a need for environmental education activities to raise public community (schools, young people and adults) to implement the protection and restoration measures of Imeretian oak tree stands. The protection and conservation the old-growth forests including damaged by stem rot or dead trees currently is considered as an important task for foresters and environmentalists (Dudley and Vallauri, 2004). In the future, the attention should be focused on the development of organized tourism in Ajameti managed reserve.

Other findings and Research Typical Phytophthora symptoms were also found on the stems of eastern (Carpinus orientalis) in Ajameti managed reserve, the blooding lesions on the ’s main stems were found on the territory of Mtirala National Park. Rhododendron spp. infected by Phytophthora species deserve special attention in these studies. Phytophthora symptoms were also observed on several trees of Juglans spp. During the field trips, the attention was also paid to a massive and intensive decline of sweet chestnut in Mtirala National Park and Kintrishi Protected Area. The fungi Cryphonectria parasitica (Murr.) was isolated from the infected branches of . For our opinion, the study of the chestnut decline phenomenon in forests tree stands in Georgia requires further deeper phytopathological and genetic researches and should be continued in this scientific direction. A very rare and interesting lichen Lobaria pulmonaria (L.) Hoffm. (Family Lobariaceae) was discovered on the territory of Mtirala National Park and Ajameti managed reserve (Figure 63). For our opinion, more attention should be paid to this lichen in future studies of these protected areas. On the branches and stems of Buxus colchica on the territory of Mtirala NP and Kintrisi PA an intensive growth of was revealed. It is considered that this phenomenon may be the cause of decline of boxwood. For our opinion, on the territory of examined protected areas it is necessary 10

to conduct specialized lichenology and bryology research during which it will be possible to discover rare lichens and . The results of this survey can be the basis for monitoring the environment of these protected areas. During the second field trip in October with aim to check sanitary condition of the planting material of Buxus colchica we visited the Tikeri nursery farm (Administration of Kobuleti, Lepl Forestry Agency of Ajara). The plantation of fast-growing poplars established on the territory of this nursery was examined and the fungus Cytospora chrysosperma were revealed on the infected branches of poplar. The recommendations of treatment measures against identified pests and pathogens were sent to the Administration of Kobuleti, Lepl Forestry Agency of Ajara.

Keywords: Protected Areas, Buxus colchica, Quercus imeretina, pests, diseases, soil phytopathogens, pathogenic fungi, great capricorn beetle, conservation activities.

11 INTRODUCTION

The assessment of forest pests and diseases in Protected Areas of Georgia has been conducted by Dr. Iryna Matsiakh from Forestry Department of National Forestry University of Ukraine (Lviv). The work has been carried out from June to December 2014. Dr. Volodymyr Kramarets, forest pathologist and entomologist at the Forestry Depertment, of National Forestry University of Ukraine (Lviv) has been voluntarily involved in carrying out of the field research, as well as in development of recommendations. We are very much grateful for his contribution and support to this complex work. The work has been carried out in the frame of the European Neighborhood and Partnership Instrument East Countries Forest Law Enforcement and Governance II Program (the “Program”). The program is aimed at putting in place improved forest governance arrangements through the effective implementation of the main priorities set out in the St. Petersburg Ministerial Declaration and Indicative Plan of Actions for the Europe and North Asia Forest Law Enforcement and Governance (ENA-FLEG) process. This Program specifically covers seven countries of the ENA Region, including six members of the European Neighborhood Policy Instrument (ENPI) – , , , Georgia, , Ukraine, and the Russian Federation. The Program supports selected pilot activities to be implemented with the active involvement of governments, civil society and the private sector. Most activities are implemented at a country level, complemented by strategically targeted sub-regional and regional actions. The Program is supported by the European Commission and other donors contributing to a special single-donor trust fund administered by the World Bank (WB). Implementation of the Program is led by the World Bank, working in partnership with the International Union for Conservation of Nature and Natural Resources (IUCN) and the World Wide Fund for Nature (WWF) (all three collectively referred to as the “Implementing Organizations” or “IO’s”) and in close coordination with governmental and nongovernmental stakeholders of the participating countries.

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1. BACKGROUND INFORMATION 1.1. Status of Biodiversity

Georgia is located between the south slope of the Caucasus Mountains, the east coast of the , and the northern edge of the Turkish Anatolia plain. The area of the territory is 697000 km2 and has a population of 490.5 thousand persons (as of first of January 2014) (Biodiversity assessment for Georgia, 2000; http://www.geostat.ge). The area covered by forests is approximately 40% of the country (2.8 million ha), largely in the Greater Caucasus Mountains (Georgia’s northern border), the Lesser Caucasus (its southern border), lowlands and foothills, and 40% is covered by agricultural land (Biodiversity assessment for Georgia, 2000; Introductory Report on Nature Conservation in Georgia, 2010). About 15% of forests are intensively used. According to data from the National Environmental Action Programme of Georgia in 2012 – 2016 (2012), Georgia’s forests are owned by the state – including an estimated 0.5 million ha of primary forests, 2.2 million ha of natural modified forests and 60,000 ha of protective artificial plantations. Moreover, average annual forest growth measures approximately 4.0 million m3. The distribution of forests is uneven and in some forested regions, their conditions are not very rich and do not reach 10%. Forested regions are presented on the Map (Figure 1).

Figure 1: Map of forest distribution in Georgia (data of National Environmental Action Programme of Georgia 2012 –2016)

There is a great diversity in the broadleaf and coniferous tree species of Georgian’s forests, and their richness, rarity and uniqueness are considered the true treasure of the country because most of these plants (97%) have a natural origin (Introductory Report on Nature Conservation in Georgia, 2010). So, in the Caucasus Mountains there can be found more than 200 plant community associations, 120 species of trees, 250 species of bushes and 4,500 species of vascular plants. Georgia’s ecosystem range is so wide that it is difficult to imagine - from alpine and subalpine meadows, lowland steppe grasslands, mountain and inland wetlands, coastal, coniferous and beech forests, mixed deciduous forests and oak woodlands to wetland forests, arid light woodlands, riparian and forest vegetation along rivers (Biodiversity assessment for Georgia, 2000). 13

All forests lands in Georgia can be divided into mountain and lowland forest with special ecological features, water regulation, soil protection and climate stabilization which influences plant and (Dolidze, 2013; Introductory Report on Nature Conservation in Georgia, 2010). Forests in mountain occupy 98% of the area and have the greatest conservation value. Forested lands are located on steep and gentle slopes. According to the data of Dolidze (2013), forest area is decreasing along the lower part of mountain slopes and in the west of Georgia up to 500 – 600 m.a.s.l., in the east of Georgia up to 700 – 800 m.a.s.l. and also in the sub Alpine region up to 1,800 – 2,500 m.a.s.l. At high mountain levels and steep slopes there are areas still covered with high forests in a natural state and not damaged by farming activity. Among forested lands, the areas are dominated by oriental beech at 42,5 % with a wood volume of 224.7 mil. m3; oak (Quercus iberica Steven ex M. Bieb., Q. macranthera Fisch. & Mey. ex Hohen., Q. imeretina Steven ex Woronow, Q. longipes Steven) 10.5% and 23.6 mil. m3; hornbeam (Carpinus caucasica Grossh.) 9,9 % and 24.7 mil. m3; sweet chestnut (Castanea sativa Mill.) 3.8% and coniferous species (fir, spruce, and pine) 455,000 ha, 17.4 % (Dolidze, 2013; Biodiversity assessment for Georgia, 2000). Other data presented by Introductory Report on Nature Conservation in Georgia show that broadleaf forests occupy 81% of forest cover while coniferous forests cover around 19%, and the species represented are mainly beech Fagus orientalis (46.6%), oak Quercus spp. (10.6%), hornbeam Carpinus caucasica (8.8%), Nordmann fir Abies nordmanniana (7%), alder Alnus barbata (5.5%), spruce Picea orientalis (4.5%), pine Pinus spp. (4%), and chestnut Castanea sativa (3.2%) (Introductory Report on Nature Conservation in Georgia, 2010). The slopes with oak-hornbeam forests with a predominance of Georgian oak (Quercus iberica), Q. hartwissiana and hornbeam (Carpinus caucasica) occupy the western part of Georgia. On the northern slopes, these types of forests are replaced by hornbeam and sweet chestnut (Castanea sativa), with beech forests dominating at higher altitudes. Beech forests (Fagus orientalis) with a mix of Caucasian fir (Abies nordmanniana) are located at 600 to 700 m and Caucasian spruce (Picea orientalis) and fir are distributed at 1,200 to 1,300 m, with subalpine forests of spruce and birch (Betula medwedewii) (Biodiversity assessment for Georgia, 2000). However, the most interesting and unique forests are places with mixed endemic broad- leaved forests in western Georgia that have developed in areas of high rainfall (2,500 mm/yr.). There are rich, floristically rare and relic species and communities from the Tertiary period. A rich understory and the presence of many vines and characterize these threatened rainforests (Biodiversity assessment for Georgia, 2000). Among relict and endemic species in Georgian forests are yew (Taxus baccata), Pterocaria fraxinifolia, Georgian hazelnut (Corylus iberica), Imeretian oak (Quercus imeretina), Zelkova carpinifolia, Pistacea mutica, Georgian (Acer iberica) etc. Probably, a total of 1,000 endemic plant species can be found there as well as 400 species of trees, of which 60 occur naturally only in Georgia, and another 43 only occur in the Caucasus region (Biodiversity assessment for Georgia, 2000). The understory of evergreen broadleaf arborescent species deserves a separate emphasis, because this forest level is formed with relict genera such as Rhododendron, Epigaea, Ruscus, Ilex, Daphne, Hedera, and Laurocerasus. More specifically: rhododendron (Rhododendron ponticum), Ungern’s rhododendron (R. ungernii), Smirnov’s rhododendron (R. smirnowii), cherry laurel (Laurocerasus officinalis), holly (Ilex colchica), Colchic holly (Ruscus colchicus), ground laurel (Epigaea gaultheroides), Colchic ivy (Hedera colchica), Daphne alboviana, D. pontica. Colchic understory is especially well developed in the South-Western Kolkheti until 2,300-2,400 m.a.s.l. The yew (Taxus baccata) and Zelkova (Zelkova carpinifolia) relict forests found in the East Georgia 14

reserves of Batsara and Babaneura are also worthy of mention. Some unique and relict areas formed by lowland oaks (e.g., Quercus imeretina) and Carpinus orientalis-Zelkova carpinifolia are distributed in East Georgia between the Alzani and Stori rivers (Biodiversity assessment for Georgia,2000; www.moe.gov.ge).

1.2. Protected Areas

Dolidze (2013) points that for main functional aims, the forests in Georgia are divided in two: Reserve forests (protected territories) 495, 900 ha (16.6%) and State farming forests fund 2,492,100 ha (83.4%). Georgia’s protected areas deserve special attention because the total area has significantly increased during the recent years (National Environmental Action Programme of Georgia, 2012). 7.3% (511,122.5 hectares) of all of Georgia’s territory was protected by law before 2011. Nevertheless, a high progress to further increase the total area of protected territories has already been made (Figure 2).

Figure 2: Map of the distribution of protected territories in Georgia (data of National Environmental Action Programme of Georgia 2012 –2016)

The process of planning for a major reorganization of the protected areas system in anticipation of a broad-based privatisation of state lands begun by the government of Georgia, with support from WWF-International, in 1990. The Soviet model of strict nature reserves were “zapovedniks” without development of alternative models, and 20 nature reserves are currently being transformed into 9 broad protected landscape areas with a variety of types of protection (Biodiversity assessment for Georgia, 2000). The Law on System of Protected Areas adopted in 1996 regulates all protected ecosystems in Georgia. Following the International Union for Conservation of Nature (IUCN) classification, the 15

Law contains references to IUCN categories and has established a system of strict natural reserves, national parks, protected landscapes, biosphere reserves and others, and determines a regime for their use and protection (Introductory Report on Nature Conservation in Georgia, 2010; Biodiversity assessment for Georgia, 2000).

In 1996, the following categories of protected areas with international criteria developed by IUCN have been accepted (Biodiversity assessment for Georgia, 2000): • State Nature Reserve, created and managed mainly for scientific research and/or wilderness protection • National Park, established and managed mainly for natural ecosystem conservation and recreation. • Natural Monument, established and managed mainly for the conservation of specific natural features • Managed Nature Reserve/Habitat and Species Management Area, established and managed mainly for conservation through management interventions • Protected Landscape/Seascape, established and managed mainly for natural/cultural landscape/seascape conservation, scenery preservation and recreation • Multiple Use Protected Area/Managed Resource Protected Area, established and managed mainly for the sustainable use of natural ecosystems and renewable natural resources. In addition, the Document prepared by the Ministry of Environmental Protection and Natural Resources of Georgia (Introductory Report on Nature Conservation in Georgia, 2010) represents and shows internationally accepted categories of protected areas based on IUCN. Categories of PAs of Georgia include: • Strict Nature Reserve/State Reserve, IUCN Category I; • National Park, IUCN Category II; • Natural Monument, IUCN Category III; • Managed Reserve/Sanctuary, IUCN Category IV; • Protected Landscape, IUCN Category V; • Multiple-Use Territories, IUCN Category VI. The Georgian government approved the National Biodiversity Strategy and Action Plan of Georgia (NBSAP) in May2014 (Resolution 343, 08 May 2014).According to the document there there are presently 14 Strict Nature Reserve (SNR), 10 National Parks (NP), 40 Natural Monuments (NM), 18 Managed Reserves/Sanctuary (MR), 2 Protected Landscapes (PL) and 2 Multiple-Use Territories. The total cover of the protected areas is 520 811.14ha (7.47% of Georgia’s territory). In the table below, the IUCN category status of the areas of further research in Georgia is listed (Table. 1) (Introductory Report on Nature Conservation in Georgia, 2010).

Table 1. IUCN category status of some of the further research areas in Georgia Establishment National category IUCN category Number Area (ha) date Strict Nature Reserve I Kintrishi 10 703 1959 National Park II Mtirala 15 698.8 2007 Managed Reserve IV Ajameti12 5 117 1946 Protected Landscape V Kintrishi 3 190 2007 12Ajameti Managed Reserve was established in 2007 in the territory of former Ajameti Reserve. 16

Moreover, in 2003 a new, Law of the Red List and the Red Book was adopted, which formed a legislative basis for the protection of endangered species, their harvesting and the rules of compilation of “The Red List” (Introductory Report on Nature Conservation in Georgia, 2010). This law obligates to assess the status of endangered species in accordance with IUCN criteria. The Commission on Endangered Species of the Academy of Sciences of Georgia conducted an evaluation of the state of flora and fauna species within the country using IUCN criteria and categories and developed a new Red List of Georgia. Presidential Order #303 approved this List on May 2nd, 2006 (Introductory Report on Nature Conservation in Georgia, 2010).

1.3. Brief description of the research areas

Colchida lowland (Kolkheti) is a very important and well-known refuge of Tertiary relict plants with special forest types (alder forest with evergreen boxwood understory) (Arabuli, 2007). The research conducted by Povarnistin (1938), Ketskhoveli (1960), and Sakhokia (1980) describe this unique Colchic wetland in Georgia, its density and structure and the amazing boxwood smell in air. However, recent literature does not give enough information about the situation in Colchida lowland, and the presence of boxwood is observed in the bottomland forest of the Black Sea coast (Arabuli, 2007). The ancient unique species Buxus colchica is saved on the terraces of river gorges, close to the steams in lower zone of Caucasus (Mosulishvili, 2005). Мtirala National Park is located in the south-western part of Caucasus, Lesser Caucasus, in particular the extreme western part of the Achara- range, on the Kobuleti-Chaqvi range, and between the Municipalities of Kobuleti, Khelvachauri and Qeda close to the Black Sea (http://apa.gov.ge/en/). The total area is 15,698.8 ha (Figure 3). Мtirala National Park is a special place where an interesting complex of tropical and subtropical plants is observed. Amongst the native plants that can be found here are Buxus colchica, Chestnut Castanea sativa, hornbeam Carpinus caucasica, Colchic capers Staphylea colchica, capers Staphylea pinnata, Rhododendron ponticum, cherry-laurel Laurocerasus officinalis, blackberry Rubus caucasicus, adler Alnus barbata, Rhamnus frangula Flangula alnus, linden Tilia caucasica, Black Sea holly Ilex colchica, colchic fig Ficus colchica, american pokeweed Phytolacca americana, Colchic nut Corylus colchica and others.

Figure 3: Location of Мtirala National Park (data of Agency of Protected Areas) 17 Kintrishi Protected Areas is situated in the Adjara Autonomous Republic (), in the charming valley where the river Kintrishi runs between the village of Tskhemvani and the Khino Mountains (Figure 4). The total area of Kintrishi Protected Areas is 13,893 ha. The altitude of the areas ranges from 300-2,500 meters above sea level (http://apa.gov.ge/en/). Kintrishi Protected Areas border with Kobuleti forest administration from the north, Shuakhevi municipality area from the east, Qeda municipality from the south and Mtirala National Park from the south-west. Kintrishi Protected Areas Administration is composed of two different types of protected areas – Strict Nature Reserve and Protected Landscape. Kintrishi Strict Nature Reserve was founded in 1959 with the aim to protect relict forest and endemic species, and the flora and fauna of Shuamta. Protected Landscape was set up in 2007 on the basis of Kintrishi Stricte Nature Reserve. The total area of the Strict Nature Reserve is 10,703 ha and the Protected Landscape amounts to 3,190 ha.

Figure 4: Location of Kintrishi Protected Areas

Ajameti Managed Reserve is located 230 km from Tbilisi and 15 km from in the districts of Baghdati and Zestaponi. The reserve is placed on the plains, and it includes parts of Ajameti, Vartsikhe and Sviri (Figure 5). In April 1928, 20 ha of Kutaisi forested area was turned into a nature reserve and the Ajameti Botanical Reserve was created in 1935 at the ground level of the Ajameti forest massif. Ajameti was founded as a Strict Nature Reserve in 1946 and according to the physical and geographical zoning of Georgia, the territory is located within the Kolkheti landscape district. Despite its small size it has a of the complex features attributable to the district concerned. It is located between the Kolkheti plain (Odishi lowlands) and West Caucasus zones (Javakhishvili, 1977). The total area of Ajameti Managed Reserve is 5117 ha, of which 4,738 ha is covered with forests. According to the report’s data of Vasil Gulisashvili Forest Institute (Report of forest pathology study, 2007) the average annual temperature is +13.2oC; absolute maximum temperature +39oC, 18 minimum is +9oC and average air humidity is 72%. Soil condition is very heavy clayey grounds, with some alluvial on small areas, and underground waters up to 5‐8 m depth (Makhatadze, 1977).

Figure 5: Location of Ajameti managed reserve

The territory of Ajameti managed reserve is non‐hill lowland with only one vertical girdle of mixed subtropical forests. The absolute dominant species is the ancient, rare and relict Imeretian Oak (Quercus imeretina) which occupies 95% of the area or 4,454 ha, mixed with Quercus iberica (Gulisashvili et. al. 1975; Gigauri, 2000; Report of forest pathology study, 2007). In Ajameti managed reserve, there are many 100-year-old oaks, and the age of some of the trees is than 250 years.In addition, there are Zelkova trees (Zelcova carpinifolia) that cover 2 ha (0.04% of the whole area) (Gigauri, 2000). 209 ha (4.45%) of the Ajameti Managed reserve is covered by Carpinus caucasica, 23 ha (0.51%) – by Carpinus orientalis. Dendroflora of Ajameti managed reserve is represented by up to 60 tree species, the thin undergrowth covered by rhododendron, medlar, hawthorn and rosehip. Studies show that in the undergrowth of Ajameti Managed reserve, the following species may be found: hazelnut Medlar medlar Crataegus pentagyna (W. et K.) black howthornCrataegus kyrtostyla Fing., C. microphylla C. Koch, Rosa canina, Ilex colchica Pojark., Thelycrania australis (CAM) K. San., Cornus mas L. Sindi, Rhododendron luteum, Smilax excelsa L., Rubus spp., Ruscus ponticus G. Wor. Following species in the strict nature reserve are rare but can be found: Stev, Fagus orientalis Lipsky, Sorbus torminalis L., Alnus barbata S.A.M., Hedera colchica C. Koch., J. helix L., Clematis orientalis L., Smilax excelsa L. Ajameti forests can be divided into the following types: 1. Forest with yellow azalea undergrowth covered 50% of the total area 2. Forest with shrubby understory - 30% of the total area 3. Forest with - blackberry understory -10% of the total area 4. Oak-hornbeam association - 5% of the total area 5. Forest with underlay of fescue -5% of the total area. In addition, the area of Imeretian oak forests with an yellow azalea (Rhododendron ponticum) undergrowth is currently 50% of the total area of Ajameti managed reserve. However, the 19 young tree stands are badly cared for in this forest , and human intervention is necessary to remove the yellow azalea to promote good quality trees. The current state of Ajameti’s oak forests is determined by the previous forest management on these areas. The presence of old growth oak forests in densely populated area is due to this area being guarded as hunting grounds for Georgian kings. Ajameti forest was one of the beloved hunting areas for Georgian kings for centuries and thus is well protected. It also was the ancestral allod of the King Solomon of Imereti (Report of forest pathology study, 2007). At the beginning of the 19th century, due to merger of the Kingdom of Imereti and Russia, the forest was handed over to the state treasury which sold it to the French traveller and merchant J. Gamba. After the of J. Gamba, his daughter sold the Ajameti forest and its territories on an auction where it was purchased by the well‐known landowner and wine manufacturer M. Onanov; Onanov built a wine and cognac factory in the village Vartsikhe. Barrels for wine and cognac were produced from the Quercus imeretina hardwood. The same material was used for producing the railway sleepers (ties) and supports for Chiatura main railway. This is why the tree stands of Quercus imeretina underwent a major exploitation in 19th century, and probably are less resistant to non‐favourable environment as well as pests and diseases (Report of forest pathology study, 2007). In the late 19th and early 20th century there was a continuous and unsystematic fellings of oak on the territory of Ajameti and Svirska cottages. On the territory of Vartsihska cottage oak plantations were cut down massively and irregularly leading to an attenuation of oak forests and to the appearance of large areas without forests (Rudnev, 1957). In the 20-30ies of the 20th century, a large number of selective loggings were conducted in Ajameti cottage, which are better called “pryiskovi loggings” where selected wood with the best technical qualities was taken out (Rudnev, 1957). In some areas in this period, clear-cutting of oak forests was carried out. Due to these fellings, Ajameti strict nature reserve was made in 1946 with the aim to preserve oak forests. However, even in the reserve, large oak trees which were damaged by great capricorn beetles (Cerambyx cerdo) were cut down and used as firewood (Rudnev, 1957). Currently, Ajameti’s forests are remnants of large areas of oak forests that were grown in the 19th century in the valley of river Rioni and on the slopes near the mountains. According to historical information presented by Rudnev (1957) and with reference to old, local residents, in the valley of the river Rioni these oak trees were of considerable size and were cut down before World War I. Because of their large size and thickness, special saws and cutting techniques were used for cutting. This led to the destruction and deforestation of large areas and Ajameti’s forest cottage is only a remnant of these forests and is of much lower quality than those stands that have already been cut down (Rudnev, 1957). Rudnev (1957) showed that in the early 19th century, the great capricorn beetle was not directly involved in the destruction of oak forests in Ajameti managed reserve. Firstly, root rot caused the deterioration of oak forests, and before the massive development of Cerambyx cerdo there the intensive economic activity has been conducted in the past, including: • thinning of forests when selective and illegal logging has been applied to reservation of the area; • a large number of roads around the reserve; • grazing leading to soil compaction and destruction of self-seeding and natural regeneration of oak; • the formation of a significant number of verdure plantations have reduced the biological sustainability of forests and as a result of cattle drives, conditions for the distribution and development of great capricorn beetles have been created. 20 The result of the logging on the current territory of Ajameti managed reserve was the emer- gence of a large number of oak trees on verdure origin. According to the data of Mahatadze (1987), Imnadze (1989) and Gigauri (2000), in the Ajameti managed reserve 70-75% of oak stands are on verdure origin. These are less resistant to unfavourable factors and often affected by root rot, which in turn contributes to the further weakening of trees and creates conditions for the development of great capricorn beetles and other xylophagous beetles.

21 2. LITERATURE REVIEW 2.1. A brief description of the physical geographical and the sanitary conditions of Buxus colchica in Мtirala National Park and Kintrishi Protected Areas

Buxus colchica (syn. B. hyrcana) Pojark is the evergreen Tertiary period relic plant in the IUCN Red List of Threatened Species (http://www.iucnredlist.org/details/full/32177/0). Geographic range of benefit: IUCN status Global Red list assessment: Category CR; Criteria String: A1a; B1b (i). Since 2006 Buxus colchica has been also included in the ‘Red list’of Georgia under the category VU, criterion A2, i.e. having a tendency of areal fragmentation and habitat loss. In addition, Buxus colchica is in the Red Data Book of the Russian Federation. Almost 10 very old mature individuals of Buxus colchica are protected in Georgia in , in Anaklia, 30 mature individuals in Kintrishi Protected Landscape and 15 mature individuals in Mtirala National Park (Matchutadze et al. 2013). Moreover, 19 mature individuals may be found in Machakhela Protected Landscape and 20 individuals in Erge protected by the local population. Geographic range of Buxus colchica is concentrated in the Caucasus from North Kolkheti in Russia to South Kolkheti in Turkey. In Russia the species could be found in Kavkazsky Zapovednik, in West Georgia in Kolkheti National Park, Mtirala National Park, Kintrishi Protected Landscape and Imereti Protected Area. It grows not only in the Protected Areas of Georgia but also in other territories. It can be found in the north eastern part of Turkey (Trabzon) and along Russian Black Sea coast (http://kraevedenie.net/2009/03/19/samshit-buxus/2/) and in Azerbaijan (Talish) (Matchutadze and Davitashvili, 2009). Buxus colchica is small-leaved and the most winter hardy of European boxwood which can withstand winter temperatures up to -10 ° C and up to 600 years but it grows very slowly, under favourable conditions, reaches the height of 15 m (sometimes 20 m) and a diameter can have at the bottom of the trunk up to 30 cm (http://kraevedenie.net/2009/03/19/samshit-buxus/2/). The Buxus colchica branches are straight, sticking out, 4-sided and green. Its leaves are almost sessile, glabrous, shiny, dark green and light green below or even yellowish, opaque, highly variable in shape. The flowers are small, greenish and usually asexually staminate in compact capitated inflorescences. The fruit is a small spherical box with processes disclosed in the maturation of the seed wings. All parts of the plant, especially the leaves, are poisonous. In order to grow Buxus colchica the carbonate soils are required or it can grow on limestone rock sand in alluvial soil. The plant communities of Buxus colchica create a favourable wet microclimate (Matchutadze I et al. 2013). The wood of the box tree is tough and strong. It can be used for ship-construction, in air and transport industry also it can be used for producing furniture. Buxus colchica fruit and foliage are usually used in traditional (folk) medicine and for decorating gardens and fences.

22

2.2. Boxwood Diseases

According to the data collected in 2004, 192900 ha of Georgia’s forests suffer from various diseases. Pathologies studies of the forests have not been conducted in the recent years (Introductory Report on Nature Conservation in Georgia, 2010) with the exception of episodic studies in some protected areas (short unpublished information). It was known that Buxus colchica is resistant to pests and diseases (http://kraevedenie.net/2009/03/19/samshit-buxus/2/) but the research observation conducted from 2009 shows another situation. The first signs of Buxus colchica damage were found in 2009. In 2008 there was a very dry summer and then rainy wet spring. It was proved that in 2010, there was the climate change and it was the driest year at the century but at the same time with a very low humidity. Because of these reasons was revealed Buxus colchica disease caused by Cylindrocladium buxicola (Gorgiladze et al. 2011; Matchutadze et al. 2013). Moreover, nowadays it is still in the great danger. Later in 2011 in Kintrishi Protected Areas and Mtirala National Park the boxwood trees were examined and leaf loss and discoloration of stem was observed. The two areas of 0.6 ha were selected for the research in Mtirala National Park where 60% of Buxus colchica dieback was found. On the third research plot 18 trees were planted but in a short time 75% of them were infected. For the third research, 0.009 ha were selected in Kintrishi Protected Areas for the investigation of boxwood trees diseases and 55-65% of damage was found (personal communication with Khatuna Tsiklauri, Agency of Protected Areas, Georgia). Moreover, the data collected in 2009-2011 in Kolkheti National Park informs about 40% of boxwood dieback and boxwood rust. Since 2011, the infected areas have become greater and the intensity of diseases is stronger and quicker. The boxwood samples had sent to Turkey, and and a new Buxus colchica pathogen was found in Georgia. In 2011, all reports in Georgia informed about a new disease that was not previously determined - box blight caused by Cylindrocladium buxicula. The dark brown spots on the leaf surface then mass loss of green colour and fall of the leaves were revealed. The process can be repeated and later lesions and dieback of branches and stem are appeared. Georgian scientists conducted the studies and provided recommendation connected with affection of Buxus colchica but this problem is still actual and it requires the further study. The research conducted by Tomiczek in 2013 (Tomiczek, 2013) shows the situation in Mtirala National Park and Sataplia Nature Reserve. On about 70% of all the box trees leaf loss, leaf spots and discoloration of stems was observed. Based on the previous studies it was claimed that it was caused by Cylindrocladium buxicola which is an invasive fungal species in Central Europe. Tomiczek described the health status of the box trees in Mtirala as extremely concerning and concluded that the most of the boxwood forests may die very soon (Tomiczek, 2013). Some measures were recommended and further research had to be carried out.

2.2.1. Shoot Blight of Boxwood – Calonectria pseudonaviculata (anamorph Cylindrocladium buxicola)

Species of Calonectria (anamorph Cylindrocladium) represent fungal plant pathogens associated with a worldwide distribution and a wide range of disease symptoms on a large number of hosts (Guarnaccia, 2012; Crous and Wingfield, 1994; Crous, 2002). These pathogens can cause cutting rot, damping-off, root rot, leaf blight and crown rot, shoot blight, stem cankers a lot of 23

ornamental plants (Schoch et al. 1999; Koike et al. 1999; Polizzi and Crous, 1999; Polizzi and Catara, 2001; Crous, 2002; Lombard et al. 2010a, b, d). Returning to the historical facts, in the first book of Cylindrocladium, by Boedjin and Reitsma (1950), seven Cylindrocladium species were represented with a Calonectria connection to one of these species. Later, in the monograph of Cylindrocladium by Crous and Wingfield (1994) the anamorph characteristics in the of Calonectria spp. were described and in the most important and latest monograph by Crous (2002), 28 Calonectria species were discovered, all were associated with Cylindrocladium anamorphs but for 18 Cylindrocladium species teleomorph states were not recognized. Recent studies show that 109 Calonectria and 96 Cylindrocladium species are recognised (Crous, 2002; Crous et al. 2004; Gadgil and Dick, 2004; Lombard et al. 2009, 2010d). Afterwards, Calonectria species were found that this genius is not only connected with diseases of ornamental plants (Guarnaccia, 2012). In the late 19th and early 20th century several authors demonstrated that Calonectria species could damage approximately 30 plant families (French and Menge, 1978; Peerally,1991a; Wiapara et al. 1996; Schoch et al.1999). Nevertheless, Crous (2002) was sure that the significance and impact of these plant pathogens have likely been underestimated and suggested that actually up to 100 and approximately 335 plant hosts can be infected by Calonectria species. Calonectria species can infect five forest plant families and the most important species of them are associated with Fabaceae (Acacia spp.), Myrtaceae (Eucalyptus spp.) and Pinaceae (Pinus spp.) (Guarnaccia, 2012). The main symptoms in forestry caused by Calonectria genius include cutting rot (Crous et al. 1991; Crous, 2002; Lombard et al. 2009, 2010d), root rot (Cox, 1953; Hodges and May 1972; Cordell and Skilling 1975; Mohanan and Sharma 1985; Crous et al. 1991; Lombard et al. 2009), leaf diseases (Cox 1953; Hodges and May 1972; Sharma et al. 1984; El-Gholl et al. 1986; Peerally, 1991b; Crous and Wingfield, 1994; Crous et al. 1998b; Schoch and Crous, 1999; Schoch et al. 1999; Park et al. 2000; Crous and Kang, 2001; Gadgil and Dick, 2004), shoot blight (Sharma et al. 1984; Crous et al. 1991; Crous and Kang, 2001), stem cankers (Cox, 1953; Sharma et al. 1984, 1985; Crous et al. 1991; Lombard et al. 2009) and damping-off diseases (Batista, 1951; Cox, 1953; Terashita and Itô, 1956; Sharma and Mohanan, 1982; Sharma et al. 1984; Crous et al. 1991; Crous, 2002; Taniguchi et al. 2008). In many cases, the majority of Cylindrocladium species cause leaf diseases, shoot blight, and lead to leaf diseases and shoot blight (Hodges and May, 1972, Sharma et al. 1985; Park et al. 2000; Crous and Kang, 2001; Crous, 2002; Old et al. 2003; Rodas et al. 2005). The majority of literature articles and researches show that box bligh is caused by Cylindrocladium buxicola. It is known that this fungus was first found in the where the disease was observed and the scientific name was given (Ivors, Prevention and Management of Boxwood Blight). However, both current names of fungus Cylindrocladium pseudonaviculatum or Calonectria pseudonaviculata refer to the same fungus. In 1998 in the UK the serious box (Buxus) foliar disease was found with dark brown spots on the leaves and black streaks on the stems and defoliation (Henricot et al. 2000). The leaf loss was identified but until now no tree death has been recorded. The new fungus Cylindrocladium was isolated from 30 `Suffruticosa' samples with the blight symptoms and it was identified as Cylindrocladium scoparium the most pathogenic species of this genus cause disease on a wide range of hosts all over the world (Peerally, 1991). In the UK, some symptoms of blight disease of boxwood were found in the nursery in Hampshire at the end of 1994 (Henricot and Culham, 2002). Henricot B. (2002) indicated the misidentification because of the morphological characters variability of the genus. In 1998 in New Zealand Cylindrocladium spathulatum was isolated from Buxus sp. showing leaf and twig blight 24

symptoms (Ridley, 1998) and Ridley didn’t rule out the possibility that it could be C. ilicicola which has a wider range than C. spathulatum including Buxus (Crous and Wingfield, 1994). But finally, the name Cylindrocladium buxicola was offered and confirmed use of the morphological characters, sequencing of the ribosomal 5.8S RNA and the flanking internal transcribed spacers (ITS), the b-tubulin gene, and the high mobility group (HMG) of the MAT2 mating type gene (Henricot and Culham, 2002). British and New Zealand isolates were compared and no fertile perithecia was obtained suggesting that C. buxicola is heterothallic species. However, the species Calonectria pseudonaviculata was discovered and described by Crous et al. (2002) a few months before Henricot and Culham (2002) who proposed the name Cylindrocladium buxicola. The study was determined by the examination of morphology and comparison of sequences of several regions of nuclear DNA of both new species and no teleomorph was obtained by mating of a single cultures on CLA. Historical facts indicate that the genera Calonectria 1867 used for sexual states, and Cylindrocladium 1892, used for the asexual states, are synonymized. Calonectria is the first name and thus has the priority. Therefore, the name Calonectria for species previously known as Cylindrocladium is listed in Lombard et al. (2010a). Scientific evidences of the spreading of Calonectria pseudonaviculata, often as Cylindrocladium buxicola, from the UK to mainland Europe are represented in several articles. It comes from the USA (Malapi-Wight et al. 2014; Ivors, Prevention and Management of Boxwood Blight) and British Columbia (Elmhirst et al. 2013). The researshes conducted in the field in August 2013 on affected Buxus sempervirens plants showed the rapid onset of disease symptoms. The boxwood blight have been found in Delaware, Maryland, New Jersey and the southeastern New York, US and in October 2011, shoot dieback and defoliation was revealed on Buxus sempervirens 'Suffruticosa' (dwarf English boxwood) and 'Green Balloon' outdoor, 10-cm pots at a wholesale nursery in Chilliwack, British Columbia, Canada. During the summer 2012 in Caspian hyrcanian forests, covering the Alborz mountain range of northern , a sudden leaf and twig blight disease of Buxus sempervirens subsp. hyrcana was found which was caused by Calonectria pseudonaviculata (Mirabolfathy et al. 2013). Based on the morphological, cultural and molecular characters the species Cylindrocladium buxicola was observed on potted box plants (Buxus sempervirens ‘Suffruticosa’) followed by a sudden and severe defoliation in a nursery located in the Como province (Lombardy, northern Italy) in spring 2008 (Saracchi et al. 2008). Furthermore, the several authors prove the distribution of Cylindrocladium buxicola around Europe (, , , ) and Turkey, Vietnam, suggesting that it has spread from the UK to mainland Europe (Pintos et al. 2009; Crepel and Inghelbrecht, 2003; Šafránková et al. 2013; Akilli et al. 2012; Crous et al. 2002; Cech et al. 2010). First published information about the discovery of Cylindrocladium buxicola in Georgia was represented in 2011 (Gorgiladze et al. 2011). In November 2010, in Mtirala National Park such symptoms as dark brown spots on the leaves, narrow blackish streaks on the stems and the defoliation of the relict species of the box Buxus colchica were observed. In addition, the latest research conducted by Gasich et al.(2013) showed the blight symptoms on Buxus colchica in ravine of Psyrtskha River (Novyi Afon, Abkhazia). The new diseases was identified as Calonectria pseudonaviculata and fungal pathogenicity to Koch's postulates was confirmed. According to the recent outbreaks in Northern Europe, in 2007 Cyl. buxicola was added to a EPPO list established by the European Parliament (www.eppo.org/quarantine/quarantine.htm), as quarantine pathogens. Hosts. It is known that the three main boxwood species Buxus sempervirens, Buxus microphylla (littleleaf boxwood) and Buxus sinica var. insularis (Korean boxwood) are the most susceptible to C. buxicola. Buxus colchica and Buxus microphylla var. japonica (Japanese 25

boxwood) are also hosts. The plant species within genera Pachysandra and Sarcococca have been reported as hosts to this fungus (Ivors K. Prevention and Management of Boxwood Blight, Extension Plant Pathologist). However, the information about the role of Pachysandra (spurge) and Sarcococca (sweetbox) is still limited and this plant has never been found with infections under natural field conditions (Ivors and LeBude, A new pest to the U.S. Ornamental Industry: The “box blight” pathogen Cylindrocladium pseudonaviculatum = Cylindrocladium buxicola.). The ability to be infected by the fungus is determined due to the plant genetics, as well as physical features of the plant such as a dense and compact leaf canopy. Symptoms. The symptoms of the box blight are: dark or light circle spots on the leaves (Figure 6), black cankers on the stem (Figure 7) and straw‐ to bronze‐coloured blighted foliage and defoliation (Ivors and LeBude, A new pest to the U.S. Ornamental Industry: The “box blight” pathogen Cylindrocladium pseudonaviculatum = Cylindrocladium buxicola.).

Figure 6: The dark or light circle spots Figure 7: Black cankers on the stem on the boxwood leaves (by Ivors K.) (by Ivors K.)

Leaf spots may blend together eventually cover the entire leaf surface. The infected stems can have lots of dark brown or black lesions, either linear or diamond‐shaped. The black streaks can be found on stems progressing from the bottom of the plant to the top. New growth continues to develop on healthy stems, and often the root systems remain healthy and intact (Ivors and LeBude, A new pest to the U.S. Ornamental Industry: The “box blight” pathogen Cylindrocladium pseudonaviculatum = Cylindrocladium buxicola.). Blighting and defoliation can occur rapidly with complete leaf loss under warm (64 to 80°F) and humid conditions. Shady conditions contribute to disease development. of the pathogen may sometimes be seen on the underside of the infected leaves. Under high humidity, white fuzzy masses comprised of a large numbers of spores, which are sometimes visible on infected stem and leaf tissue with the naked eye or with a hand‐lens. However, these fuzzy masses are not often observed if the environmental conditions are not appropriate right (i.e. if relative humidity or temperatures are too low). The most boxwood cuttings are propagated in humid chambers or shaded structures and liners often grow in shade; conditions that promote disease development. It is very important to know that the infected boxwood stems may remain green under the outer bark until a secondary invader or opportunistic pathogen attacks this tissue and eventually kills the plant. Disease biology. The disease cycle of Calonectria pseudonaviculata can be completed in one week. Infection occurs very quickly in warm (64 to 77°F) and humid conditions and with the help of free water. The mycelium of fungus can penetrate the leaf through the cuticle or enter through leaf stomata, is also survives on fallen leaves, and can produce spores when environmental 26

conditions become suitable. The pathogen can survive at least 5 years by remaining on decomposing fallen leaves of Buxus sempervirens. For the penetration, the fungus does not require a lesion to infect and it can enter the leaf through stomata and high level of humidity of free water on plant tissue are not necessary for successful (Douglas, Boxwood blight – A new disease for Connecticut and the U.S.) The pathogen may be distributed at a short distance by water (splash dispersal), contaminated tools, birds or other and at a long distance by wind. Human activities, such as pruning, may also spread the fungus (Ivors and LeBude, A new pest to the U.S. Ornamental Industry: The “box blight” pathogen Cylindrocladium pseudonaviculatum = Cylindrocladium buxicola.).

2.2.2 Other Boxwood Diseases

Volutella box blight - Volutella buxi (teleomorph Pseudonectria rouselliana) The infected boxwood stems may remain green under the outer bark until secondary opportunistic pathogens attack this tissue and eventually may kill the plant. The fungus Volutella buxi is often associated with boxwood blight pathogen as secondary cancer pathogen and is known to cause Volutella blight, but it does not cause the box blight on its own (Ivors, Prevention and Management of Boxwood Blight; Ctrouts and Winter, 1994). Voluttela blight symptoms usually appear in spring, as individual shoots or entire plants exhibit poor growth (Douglas, Boxwood blight – A new disease for Connecticut and the U.S.). Volutella buxi can be distinguished from box blight easier because it produces masses salmon-coloured spores on the undersurfaces of infected leaves that are visible. The bark of infected branches may be peeled and darkened and discoloured (Douglas, Boxwood blight – A new disease for Connecticut and the U.S.). A very important feature is that the fungus does not cause black circle spots on the leaves and mat infects only one branch of plant whereas boxwood blight develops on the bottom and gradually moves upwards on the stems often making the plant look “top heavy” and leaving foliage only at the branch tips (Ivors, Prevention and Management of Boxwood Blight).

Macrophoma leaf spot – Macrophoma candolleri Macrophoma candolleri is considered to be a secondary pathogen of Buxus spp. that causes raised small black spots on the undersurfaces of dying leaves (Ivors, Prevention and Management of Boxwood Blight). The main disease diagnostics include change of the color of the leaves to yellow-straw and the influence of several other factors, such as and environmental conditions (winter, frost, drought) (Douglas, Boxwood blight – A new disease for Connecticut and the U.S.). The presence of Macrophoma candolleri indicates that the plant is damaged by some other factors and any recommendations are developed but for predisposing factors which should be addressed (Hansen, Major Diseases of Boxwood). Moreover, the boxwood can be damaged by winter or sunscald and the results of these injuries are associated with dieback of leaves, twigs and sometimes-even plants. Under the influence of the different weather conditions leaves may start to reddish, may become brownish and bronze, the bark of the stems and branches may be peeled and later diebacked. Analogously, this known disease complex is called “boxwood decline” and it includes nematodes (Meliodogyne and Pratylenchus) and fungus Clonostachys buxi (=Paecilomyces buxi =Verticillium buxi) which may contribute to this complex. This complex may influence the boxwood decline for several years this influence is manifested by some insignificant defoliation, stunting, wilting, loss of the vigor and chlorosis (Douglas, Boxwood blight – A new disease for Connecticut and the U.S.). 27

Phytophtora Root Rot According to the data of the USA Department, even the boxwood has suffered from several defoliations and has been infected by the box blight but the root systems remained healthy, intact and without damages (Ivors, Prevention and Management of Boxwood Blight). Nevertheless, the roots infected by Phytophthora may look different. Phytophthora spp. has been identified as causative agents of dangerous diseases of agricultural, ornamental and forest plants (et al. ). The special concern is due to the rate of Phytophthoras spread, a wide range of host plants and the lack of effective control measures. The research recently conducted shows that the cause of the infections of different plants in different parts of the world is often the same aggressive pathogenic Phytophthora species (Matsiakh, 2013). is one of the most famous group of the pathogenic organisms that affects the forest ecosystem, wood and ornamental plants (et al. ). Until recently, this class belonged to Fungi Kingdom, but contemporary genetic and biochemical studies have shown a significant difference between them (et al. The genus Phytophthora has a complicated family history research because for a long period there were different opinions on the expediency of combining the genus Phytophthora and the genus Pythium. However, in the late 20th and early 21th century the undeniable uniqueness of this genus has been proven by using the molecular taxonomy methods (et al. Matsiakh, 2013). The species of the genus Phytophthora (from Greek: φυτόν (phyton) - "plant" and φθορά (phthora), "destruction" - "destroyer of plants") are primary parasites of fine roots causing the root rot and the bark lesions of young shoots and stems, as well as mature trees and of many species (). Phytophthoras influence includes some visible symptoms on the trees such as thinning of the crown, delay of the plant growth and drying of lateral branches. The leaves may become abnormally small and change their color to straw-yellow and green. Over the time, the branches and crowns of trees become completely dead. Species of the genus Phytophthora can be airborn, soilborn or waterborn (Erwin and Ribeiro, 1996). First ones have an advantage of transferring their zoospores through air, but they can survive in soil too. The biological cycle of the second group is connected with soil and most of these organisms are fine root pathogens infecting plant tissues via zoospores especially when the soil is saturated with water. The third group of Phytophthora is linked to the water ecosystems and its life cycles, as well as their sexual behavior. They can be transferred from tree to tree when direct contact of zoospores with the bark tissue take place (Oszako, 2005). Significant precipitation, the increase of soil moisture and optimal temperatures cause favor its development and further spread. In addition, structures such as oospores and chlamydospores survive in infected seedlings and root rhizosphere in the nursery, they stick to the tools, wheels of the cars, tractors and people’s shoes. One of the main pathways of Phytophthora spreading is water carrying spores. The first record of such a spread dates back to 1921, when Phytophthora cryptogea was found in water used for watering the plants in a nursery greenhouse (Bewley and Buddin, 1921). Since the 19th century more than 20 species of Phytophthora and representatives of the genus of Pythium, Fusarium, Rhizoctonia have been isolated from water (Hong, 2005; Themann, 2002). Today all over the world, the spread of these pathogens is becoming stronger and faster. Venediyapina (1985a) gave the first information about Phytophthora disease in Abkhazia in 1985. It was the discovery of Phytophthora cinnamomi in soils of chestnut woods of Abkhazia. According to the study of Hasen Mary Ann (Major Diseases of Boxwood http://www.mastergardenproducts.com/gardenerscorner/boxwooddisease.htm), Buxus sempervirens cv. 'Suffruticosa' and B. sempervirens cv. 'Arborescens' are susceptible to 28

Phytophthora disease, which is caused by Phytophthora parasitica. In addition, this disease was found in Virginia on Buxus microphylla. American Boxwood Company shows that Phytophthora is a plant destroyer and it gives some information how serious a problem can be (American Boxwood Company Boxwood Diseases http://www.americanboxwood.com/index.php/plantcare/56-boxwood-diseases) and that a soil-borne fungus can affect all cultivars of B. sempervirens at any age or size. Phytophthora parasitica can damage root, stems and leaves. Infection may begin in wet and cool soil about 58oF to 70oF (14oC to 21oC), usually in spring and autumn. The progress of the disease and first damage appears when the temperature of the soil is higher and then it occurs at 75oF (24oC). The greatest effect is observed at 85oF (29oC). When Phytophthora infects the boxwood, it seldom survives. Produced spores in the water that flows in the soil, Phytophthoras can damage the boxwood growing in poorly drained soils very well. The research in hardy ornamental nursery stocks with Buxus sempervirens had been conducting for three years in Poland (Orlikowski et al. 2008). The first symptom of disease was yellowing of individual stems spreading slowly to other shoots. Strongly invaded stems were straw coloured. The bark started to separate from the wood which turned bluish. Phytophthora citricola was determined as a new pathogen on Buxus sempervirens in Polish ornamental nursery stocks. At this time in spring of 2008, some gradual decline was revealed on 6-year-old Buxus rotundifolia plants in a garden in central Italy (Vettraino et al. 2010.). Among 150 boxwood plants, 70% of them had symptomatic damage and 25% among them were completely wilted. The symptoms of disease included stunting, reduced growth, leaf chlorosis when leaves were light green at first and then became yellow, bronze or straw coloured and necrotic bark lesions at the base of the stem. First foliar symptoms were observed only on several branches and later it extended to the whole crown. After using immunological field tests (Pocket Diagnostic, CSL Diagnostics, Milan, Italy) and the isolation on PARHP (2) from necrotic tissues, P. citrophthora was determined (Vettraino et al. 2010). Moreover, at the end of September in 2012 in the commercial nurseries in Italy the collar and root rot associated with the severe wilting and desiccation of foliage on Buxus sempervirens was discovered (Luongo et al. 2013). About two hundred boxwood plants were detected and 50% of them showed the change from green to straw colour and diffused desiccation resulting in 20% mortality. The leaves symptoms were the same as on Buxus rotundifolia described above. Phytophthora nicotianae was detected by plating small pieces of stem and root tissues on P5ARPH selective medium, from soil samples baited with azalea and camellia leaves and using DNA analysis. Symptoms. Phytophthoras symptoms are very specific because they include visible and underground features. Aboveground symptoms have gradual signs such as poor growth, off-color foliage, wave edges and changing colour of leaves from light green to yellow, bronze and straw- color. The leaf symptoms may appear on just a few branches or on the entire plant, depending on the extension of the infection of the roots and they can remain attached to the twigs. The bark of the infected boxwoods dies and can be easily removed from the stem. The reduction of functioning roots is preceded by yellowing and the death of the plant top. Aboveground symptoms of the stem include dark brown and black coloration of the vascular tissues under the bark at a ground level to a few inches (7 cm to 18 cm) above and this dark colour is a response of the vascular tissue to the fungus. The internal symptoms of stem become apparent in partial or complete blockage of nutrient and water movement in the stem (American Boxwood Company Boxwood Diseases http://www.americanboxwood.com/index.php/plantcare/56-boxwood-diseases). 29

There is a very important feature when foliar symptoms are visible on the plant, roots may be in brown colour and complete dull whereas healthy roots have a bright light tan colour. Disease features. Phytophthora diseases may be described as a slow progressive process that may last for a long period generally occurring in large plants of 20 years old or more. Nevertheless, Phytophthora root rot prefers a wet soil (a large amount of moisture 70%) and the temperature of about 20 degrees. At this temperature sporangia form and the mass floating zoospores mature. They have the ability to mix in the soil, to touch the most sensitive roots, surround and grow into the tissue forming penetrable hyphae. This process is ideal in peaty soil with high level of humidity (Łabanowski et al. 2000.).

2.3. Boxwood Pests

The state of boxwood can also be influenced by the development of certain species of insect-phytophagous. The threat of boxwood green spaces and natural populations may be represented by the superfamily Coccoidea (Hemiptera): Lepidosaphes ulmi (L.), Parthenolecanium corni (Bouché), Eriococcus buxi Fonsc. (Łabanowski et al. 2000). Еriococcus buxi is the most dangerous among these species. It characterizes the narrow specialization of feed and inhabits the branches and leaves of Buxus species. During the year, two generations of this pest may develop. The first generation can last from early May to November, the second from August to May or June next year. The larvae of first or second age overwinter. After its appearance, the young female covered with a waxy bloom, which eventually turns into a felted bag where the female lays the eggs. Mass reproduction and nutrition of Eriococcus buxi is accompanied by yellowing and shedding of the leaves on individual branches first, and then the whole bush that leads to significant damage and dieback in boxwood green spaces (Yatskova, 2012). The other two species Lepidosaphes ulmi and Parthenolecanium corni are characterized as a wider feeding specialization and may inhabit the different trees and shrubs but can also damage the boxwood (Łabanowski et al. 2000).

Insect-filophagus of boxwood The Monarthropalpus flavus may develop on the leaves of the ornamental boxwood plants. Females lay eggs on the surface of the leaf; the larvae bite in the middle and form a shell overwinter. The damaged leaves are slightly deformed and swollen and are not noticed immediately. Then leaves turn yellow, cover with spots and fall. This leaf miner pets can seriously damage the leaves and lead to the death of boxwood bushes (Kluepfel and Scott, 2004).

Cydalima perspectalis The new threat of natural boxwood in Colchida lowland caused by the dangerous invasive species phyllophage Cydalima perspectalis (Walker, 1859) [= Glyphodes perspectalis (Walker, 1859)] (: Srambidae: Pyraustinae) in the Caucasus has recently discovered. According to the data of Russian experts, the caterpillars of this butterfly was found on September 22 in 2012 in Sochi on boxwood bushes in temporary nursery where the planting material was imported from Italy for the planting area of the Basic Olympic Village. The conducted control measures failed and pest spread to the green stands of Sochi and later entered the natural grouping of boxwood on the territory of yew-box grove in Caucasian Biosphere Reserve (short unpublished information in Georgian; Eskin and Bibin, 2014). 30

Nowadays, the danger is the rapid development of this invasive species. Depending on the climatic characteristics Cydalima perspectalis can produce two or four generations. The following features (Gutue et al. 2014; Eskin and Bibin, 2014; News…, 2013) can cure the damage of boxwood: • the dark green hairy caterpillars with black heads feed on the leaves (at the end their body’s length may reach up to 4 cm); • the leaves and stems are covered with silk threads in which excrements and larvae skins and black head capsules of caterpillars are visible; • the larvae leads to the rapid decline of boxwood (for 7-14 days). In Europe and the Black Sea coast Cydalima perspectalis damages different species of boxwood but at home (Asia) caterpillars feed on leaves of Japanese spindle ( japonicus Thunb.) and winged spindle (Euonymus alatus (Thunb.) Siebold) and on leaves of holly purple (Ilex purpurea Hassk.) as well (Eskin and Bibin, 2014).

2. 2. A brief description of the physical‐geographical and the sanitary conditions of Imeretian Oak in Ajameti managed reserve

Imeretian oak (Quercus imeretina Steven ex Woronow) is a protected relict Tertiary period tree in Ajameti managed reserve which is listed in the IUCN Red List (Category - Vulnerable B1 + 2c ver 2.3). Moreover, Quercus imeretina is rare endemic tree of Colchida lowland (Kolkheti) in western Georgia and on the small places along the Black Sea in Russia (The IUCN Red Lis, 1998). Menytskiy Y. (Menytskiy, 1971), who was examining the morphology of Imeretian oak, came to the conclusion that it is probably a subspecies of L. subsp. imeretina (Steven ex Woronow) Menitsky and nowadays it is recognized by the majority of dendrologist (Taxonomic Nomenclature Checker). Quercus imeretina is an endemic tree species of West Georgia that used to occupy the large areas on the right bank of the river Rioni. However, the impact of anthropogenic factors (deforestation because of agricultural activities and cutting down trees for timber and fuel, etc.) led to a reduction of Quercus imeretina areas (Nakhutsrishvili, 2013). According to the data of Gulisashvili V. and co-authors (Gulisashvili et al. 1975) Imeretian oak forests grow only in the eastern part of West Georgia on a heavy and clay soil with deep level of groundwater (6-8 m and deeper). This region has a soft maritime climate and high precipitation throughout the year, which to some extent compensates depth of groundwater. Climate is conducive to frequent repetition of productive years and the acorns begin to germinate in the autumn (Gulisashvili et al. 1975). The composition tree forest stands also includes Georgian oak (Quercus iberica), Zelkova trees (Zelcova carpinifolia) and Oriental Hornbeam Carpinus orientalis Mill. Imeretian oak can also form the monodominant tree stands. Singly or in groups Imeretian oak appears in the forests of the northwestern part of Colchida lowland. These authors distinguish six types of tree forests stands with Imeretian oak: Quercetum imeret.-azaleosum; Quercetum imeret.- carpinuleto-crataegosum; Quercetum imeret.-hypericosum; Quercetum imeret.-muscosum; Quercetum imeret.-graminosum; Quercetum imeret.-ruscosum. They prove that Imeretian oak may be the best naturally renewed in conditions of Quercetum imeret.-carpinuleto-crataegosum, Quercetum imeret.-hypericosum and Quercetum imeret.-ruscosum (Gulisashvili et al. 1975). Dolukhanov (2010) offers a different classification of forest types with Q. imeretina in Georgia: 1) Moist oak forests with Smilax excelsa; 2) Fresh oak forests with Rhododendron luteum; 31

3) Dry oak forests with Carpinus orientalis; 4) Dry oak forests with Ruscus colchicus; 5) Dry oak forests with Hypericum xylosteifolium; 6) Dry oak forests with Zelkova carpinifolia. It is known that Quercus imeretina belongs to the Quercus family of and is spread only in Georgia especially its western part, can be met in Abkhazia, Lower Svaneti, Racha‐Lechkhumi, Samegrelo, Imereti, and Ajara; as relatively significant woodland it is represented only in Imereti region (Report on forest pathology study of Ajameti Strict Nature Reserve, 2007). Quercus imeretina can grow as a tree of first magnitude and morphological features of the tree are very specific such as stalkless (sessile) leaves and grape‐type fruit (acorn). This tree grows only in lowlands and at the height of 300‐400 m above the sea level. Moreover, Quercus imeretina has a long vegetation period, requires a great demand for air in soil, and high air humidity of place where it grows with a large number of annual precipitation and it conditions like warm and light. As in Kolkheti lowlands moist soil dominates Imeretian oak adapts well to the long‐term summer draughts at the places, does not grow well on bad non‐developed, macadam and detritus lands and grows well on the clayey soil (Report on forest pathology study of Ajameti Strict Nature Reserve, 2007). This tree characterizes a strong and deep root system that deeply penetrates the soil and on the moist grounds a well-developed horizontal roots system is noticeable. The fruits of Imeretian oak appear in 10-15 years, the wood of Quercus imeretina is firmness and beautiful with brown‐yellow core and narrow aluminous layer. In the tree stands composition in Ajameti Georgian oak (Quercus iberica) M.Bieb may be found. Morphological features of this oak give the reason to believe that it geographically similar to the sessile oak (Matt.) Liebl. subsp. iberica (Steven ex M. Bieb.) Krassiln. (Menytskyy, 1971) (Taxonomic Nomenclature Checker). In addition, Georgian oak has a larger growing area and the forests where it grows are in the Caucasus (Georgia, Armenia, Azerbaijan), North Turkey and sometimes as part of plants it occurs in the far of Moldova and . The forest belt of Georgian oak extends from 500-600 to 1000-1100 m above the sea level, but the orographic features of the area can make some adjustments, particularly in western Georgia (in the rivers Rioni, Inguri, etc.). Georgian oak rises to 1750- 1800 m above the sea level which forms mixed tree stands with Caucasian oak or Persian oak () Fisch. & C.A. Mey. Ex Hohen. and occurs in the second pine forest stands (Gulisashvili et al. 1975). Quercus iberica as Quercus imeretina belongs to the family of Fagaceae, but differs in the leaves which have stalks and its fruit (acorn) is either situated in the cuplike base or has a very short stalk (Report on forest pathology study of Ajameti Strict Nature Reserve, 2007). Quercus iberica mainly grows in the South Caucasus, partly in the North Caucasus and North Iran. It also strives to light and warmth and can grow in lower front side of mountainous forests about 1000 m above the sea level, thus creating oak woodland band; however, at some places it can also grow at 1400‐1500 m above the sea level. Another characteristic of Quercus iberica is the ability to grow on almost any type of land without any special demands for the soil. As it avoids over‐moist and moist soil, it was only found in mixed tree stands with Quercus imeretina on well‐drained lands in Ajameti managed reserve. Quercus iberica does not grow on heavy clayey soil and propagates by means of the seeds as well as through vegetation – stub seedling. Quercus iberica is a heavy hardwood trees with high technical features and is widely used in furniture and construction industry (Report on forest pathology study of Ajameti Strict Nature Reserve, 2007). 32

It was discovered that the species of oak trees do not differ in annual fruiting and usual fruiting is observed in one year. The average duration of the growing season ranges from 220 days to 240 days and depends on the early or late spring. In 2012, the fruiting of Imeretian (Georgian) oaks and Zelkova trees was lower then average level. Zelkova carpinifolia (Pall.) K.Koch is a relic tree of the Tertiary period and is also included in the IUCN Red List Category - Lower Risk / near threatened. In addition to Georgia (Colchida lowland), the forests stands with Zelkova carpinifolia grow on the territory of Armenia, Azerbaijan, Iran, Turkey (The IUCN Red List, 1998). There are other IUCN Red List relict tree species on the territory of Ajameti managed reserve: Caucasian wingnut (Pterocarya pterocarpa (Michaux) Kunth ex Iljinsk – Category: Lower Risk/least concern (The IUCN Red List, 1998) and Red List of Georgia: Pastuchov's ivy (Hedera pastuchowii Woronow) and Turkish (Corylus colurna L.).

2.4. Imeretian Oak Diseases and Pets

The process of oak forests dieback (decline) has been the focus of foresters for more than 100 years. The cases of oaks dieback in Europe have been observed occasionally since 70s of the XIXth century (Rykowski, 1997). Researchers often agree with the idea that dieback oak effect is caused by complex factors, including the impact of extreme weather conditions, changes in the hydrological regime, damage of the oak phytophagy and disease, human impact etc. (Osipov et al. 1989; Tsaralunha, 2002, 2005; Kätzel et al. 2006; Selochnik, 2008). A certain role in the process of degradation of the oak forests belongs to the disease, which play a secondary role (Selochnik, 1998, 1999, 2002). An intensive decline of oak forests is connected to the extreme climatic conditions including very high or vice versa low temperatures, the influence of long-term drought or changes in the level of groundwater, etc. (Siwecki, 1991; Ważny et al. 1991; Bernadzki and Grynkiewicz, 2006). In addition, oak decline is sharply activated by a massive development of leaves insects and pathogens after dry periods (Stocki, 2005). The defoliation of trees plays a very important role that dramatically impairs their physiological state (Blank and Hartman, 2004). One of the reasons that significantly affects the oak forests deterioration is a long coppice restoration of this species in the large areas that leads to a significant weakening of forest stands and reduce their resistance to abiotic and biotic factors (Kalynychenko, 2000; Tsaralunha, 2003, 2005). However, the high stability of oak forests to extreme situations was discovered. So, even after the catastrophic damage of the tree stands (in Shypovyj forest and Voronezh forest reserve) the conditions of the trees were improved during the periods conducive for their existence (Osipov et al. 1989). Oak decline disease was first detected in Georgia in the 1970s and was associated with Verticillium sp., Rhinotrichum spp., Erwinia multivora and E. lignifilla on Quercus imeretina, Armillaria mellea on Q. iberica, and leaf mining insects (green oak roller moth (Tortrix viridana), winter moth (Operophtera brumata), oak umber (Erannis leucophaearia) were found on both Quercus spp. (Imnadze et al. 1989) According to the data of the Report on forest pathology study of Ajameti managed reserve (2007), the first facts of wilting of Quercus imeretina were registered in 70ies of the XXth century, which reached its peak in the middle of 80ies and was caused by the leaf‐eating pests (Operoptera brumata L., Erannis difoliaria, Ocneria dispar). 33

During examination of the oak forest stands in Ajameti managed reserve in 2007 by the researches of Vasil Gulisashvili Forestry Institute 12 species of pathogenic fungi and 13 species of pests were found. Among them, were identifined Cerambyx cerdo acuminatus which damages trees and Ganoderma applanatum which penetrates through outlet openings of beetle adults and leads to stem (komlevoyi) rotten wood (Report on forest pathology, 2007). The information about negative impact of climate anomalies on the resistance of coppice shoots of oak stands was indicated in this report. The significant temperature decrease from ‐1 to ‐4oC was recorded at the end of April in 1980, 1983, 1984 and 1987 and probably it may have influenced the deterioration of forest conditions (Report on forest pathology, 2007). In Ajameti managed reserve the oak trees have been damaged by great capricorn beetle for a long time. There are several subspecies of great capricorn beetle, including Cerambyx cerdo cerdo Linnaeus (listed in the Red List in many European countries) and Cerambyx cerdo acuminatus Motschulsky, which is more common in the Caucasus and is protected in Armenia (The Red Book of Animals of the Republic Armenia, 2010). Some researchers (Sama, 2002; Miroshnikov, 2009) indicate the groundlessness of selection subspecies Cerambyx cerdo acuminatus. Some papers note that the distribution of this pest was caused by improper management and industry over the second half of XIXth century in Ajameti oak stands and according to the data of Supatashvili et al. (1967), by 50ies of the XXth century the number of trees occupied by Cerambyx cerdo acuminatus was about 13%. Rudnev (1957) showed that the population of great capricorn beetle in Ajameti managed reserve ranged from 9% (in the middle of forest) to 29-42% in the areas near the village. A survey conducted in 2007 indicated that Cerambyx cerdo acuminatus may widely spread within the oak stands and since that 20% of forest has been damaged (Report on forest pathology, 2007). The biological features of great capricorn beetle were studied in great details (Rudnev, 1957; Lozovoy, 1958; Sama, 2002). However, so far there is no consensus about trees where larvae of Cerambyx cerdo can develop. Rudnev (1957), who studied the development of Cerambyx cerdo in the forests of Ukraine, Abkhazia and Georgia, considered that Cerambyx cerdo is only populated on live but weakened trees and stumps of fresh oaks. Other tree species, in his opinion, are very rarely inhabited. Sama and Lozovoy (2002) who studied this species in the forests and parks of Georgia agreed with this opinion (Lozovoy 1958, 1965). However, many authors believe that the great capricorn beetle is feature of the wider stern specialization. According to the data of Samedov (1963) in Azerbaijan Cerambyx cerdo often appears in orchards and deciduous forests. Miroshnikov (2009) and Albert (2012) indicate a wide range of forage species of Cerambyx cerdo. Almost all researchers indicate the ability of Cerambyx cerdo to colonize alive but weakening oaks with dry tops, with mechanical damage of stem and stem rot. The physiological condition of the tree plays a decisive role in its settlement by great capricorn beetle (Rudnev, 1957; Albert et al. 2012). In the literature, Cerambyx cerdo is considered to be as a physiological and technical pest. At the beginning the first year larvae feeds itself in the thickness of bark only on alive but weakened tree, then they move under the bark deep into the wood where make feeding road in length from 40- 60 to 100 cm even next years (Lozovoy, 1958; Sama, 2002). Pupa forms in the middle or at the end of summer. Adults appear in late summer or early autumn and winter in the pupa places. The imago-flying period lasts from May to late summer and Cerambyx cerdo has three-year generations (Rudnev, 1957; Miroshnikov, 2009). Morphological features of great capricorn beetle is very well studied (Rudnev, 1957). The body length of adults range from 23 to 65 mm. The antennas of males are 1.4-1.7 times longer than the length of body; the antennas of females are equal to the length of elytra (sometimes a little 34

longer or shorter). In order to get some additional feed, the beetles may drink juice that comes out of the places damaged by larvae. The larva is white and yellow and at older age can reach the length up to 100 mm, width to 18 mm. The deterioration of decline process in the oak forests is also contributed by pathogens. However, the majority of researchers believe that the fungus only accelerate the process of dieback of the oak forests and may be as satellites of other destabilizing factors (Yakovlev, 2007). T. Jung shows how in Europe several species of Phytophthora citricola, Phytophthora cactorum, Phytophthora cambivora, Phytophthora quercina, Phytophthora alni and Phytophthora pseudosyringae are causing root and collar rots, and aerial, bleeding bark cankers on stems and branches of many tree species such as European beech (Fagus sylvatica), (Acer platanoides and A. pseudoplatanus), horse chestnut (Aesculus hippocastanum), sweet chestnut (Castanea sativa), limes (Tilia spp.), White fir (Abies alba), spruce (Picea abies), scots pine (Pinus silvestris) and several oak (Quercus robur, Q. petraea, Q. cerris, Q. ilex, Q. frainetto, Q. pubescens) and alder species (Alnus glutinosa, A. incana, A. cordata, A. viridis) (Jung, www.baumkrankheiten.com/.../phytophthor). Between 1993 and 2004, more than 150 oak stands were investigated in twelve European countries and it was shown that on a broad range of site conditions Phytophthora quercina and nine other soilborne Phytophthora species are strongly involved in the etiology of European oak decline by causing a progressive destruction of the fine root system (Jung, www.baumkrankheiten.com/.../phytophthor).

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3. Methodology 3. 1. Field Research

The field research was conducted in June and October 2014 on the territories of examined Protected Areas. The sanitary assessment of tree stands and affected trees was carried out, the soil samples and samples of fruiting bodies of fungi were collected and after studying the found larvae and adults the species compositions of insect phytophagous were determined. The pictures and photos of the next chapters are personally taken by the executor of these researches. The study of the sanitary condition of forest plantations was conducted using the forest- pathological examination (Vorontsov et al. 1991). The samples of fruiting bodies, damages and adults or larvae for studying the species composition of pathogens and pets were collected and determined using the special literature and atlases (Atlas ..., 1999; Gusev, 1984; Bondartseva and Parmasto, 1986; Bondartseva, 1998; Łakomy and Kwaśna, 2008; Hartmann et al. 2009; Kolk and Starzyk, 2009). Latin names of fungi and pathogens and abbreviated names of their authors were specified using the site http://www.mycobank.org. For establishing the features of distribution of great capricorn beetle and the sanitary condition of Imeretian oak in Ajameti managed reserve, the trees enumeration was conducted on running lines. These lines had been built in the stands of different age and degree of human impact and the account the number of paths and roads, places of grazing were taken into. The diameters for each trees and their characteristics were measured using IUFRO tree classification (Table 2) (Leibundgut, 1958). Table 2. IUFRO tree classification Criteria Class Number Significance 100 Trees in the upper layer; their height is higher than 2/3 of dominant height 200 Trees in the middle layer; the height is Height 1/3-2/3 of dominant height 300 Trees in the lower layer; the height is Positional less than 1/3 of dominant height 10 Vigorous trees (with vigorous growth) Vitality (health state 20 Normal trees (with normal growth) and vigour) 30 Small trees (with low growth) 1 Trees tending to evolve Hierarchical trend 2 Trees not modifying the social position 3 Trees tending to regress 400 Main (principal, elite) trees; by quality, growth potential, other qualities, they Silvicultural value should be favoured 500 Useful secondary trees; helping the main trees 600 Damaging secondary trees; competing with the main trees Economic 40 Trees with boles of good quality; at least 50% of bole volume is valuable (of high Quality of bole (trunk) quality) 50 Trees with boles of normal quality; at least 50% of bole volume is of moderate quality 36

60 Trees with boles of low quality; less than 50% of bole volume is of moderate quality 4 Trees with long crowns; crown length more than ½ of total tree height Crown length 5 Trees with moderate crowns; crown length between ¼ and ½ of total tree height 6 Trees with short crowns; crown length less than ¼ of total tree height

The diseases and damages of tree stands were studied following recommendations of Rudnev (1957), Tsaralunha and Kaharmanova (2006), Furmenkova (2009), Sanitary rules… (1995). The following features were pointed out: • the condition of the crown (top and degree of decline the main branches); • an extent of the crown damaged by phytophagy insects; • the condition of the trunk and the presence of frost cracks, traces of lightning, stripped of bark, hollows, cancers and lesions, fruiting bodies etc.; • the tree settlement by great capricorn beetle and other insects-ksylophagy. The results of the survey were used for establishing the pathological condition of Imeretian oak in areas with different levels of disturbance and for analysis features for distributing great capricorn beetle in oak stands in Ajameti managed reserve.

3. 2. Laboratory Work

The processing and the preparation of the collected samples were carried out in the Laboratory of Forest Protection of Forestry University of Ukraine for further laboratory analysis. The soil and tissues samples of symptomatic Buxus colchica and Quercus imeretina plants were collected during the field trip at the beginning of June in 2014 and analyzed in Italy (DIBAF University of Tuscia, Viterbo) and in Poland (Forest Research institute, Warsaw) using different approaches. Small pieces of symptomatic tissues were surface-disinfected and placed on aqueous agar (distilled water and 1.5% agar-agar) supplemented with antibiotic streptomycin (3 mg/100 mL) in petri plates and incubated until fungal growth will initiate. Pure cultures, obtained by the tips of the fungal hyphae were maintained on PDA and examined for colony morphology. Isolates were identified based on morphological characteristic of the micro and macro structures and the growth rates at different temperatures. For the molecular identification the entire ITS region was amplified using the ITS1f/ITS4 primers. As an underlying basis to identify the fungi, the sequences was manually edited and compared with available data from GenBank databases (National Centre for Biotechnology Information website; http://www.ncbi.nlm.nih.gov/) using the BLAST program. Small pieces of symptomatic tissues were surface-disinfected and placed on PARPH agar (Tsao and Guy 1977) (Figure 8). Sporangia production was performed into sterile distilled water (ca. 20 mL) or 10% soil solution. Isolates were characterized morphologically with published keys for genus Phytophthora (Waterhouse 1963, Stamps et al. 1990, Erwin and Ribeiro, 1996).

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Figure 8: Laboratory work

Phytophthoras were isolated from soil samples using the baiting technique (Figure 9). Young leaves from healthy different hosts (e.g. oaks, beech) and carnation petals were used as baits according to Jung et al. (1996). Infected leaflets were cut into small pieces and were plated onto selective PARPNH agar. Isolates were identified as described above.

Figure 9: The baiting technique for Phytophthoras isolation

DNA isolation and amplification. DNA extraction from soil was carried out using the Power Soil® DNA isolation kit (MoBio Laboratories Inc, CA, USA) according to the manufacturer’s protocols. To perform metagenomic analysis, genomic DNA was amplified using the tagged primer pair ITS6 (5- AxxxGAAGGTGAAGTCGTAACAAGG-3) and ITS7 (5- BxxxAGCGTTCTTCATCGATGTGC-3), where A and B represent the two primers 38

(CGTATCGCCTCCCTCGCGCCATCAG and CTATGCGCCTTGCCAGCCCGCTCAG) and xxx represents the barcoding key (DNA TAG). Extraction of genomic DNA from mycelium was performed as reported by Lee and Taylor (1990). The PCR was carried out using the ITS1-ITS4 primers (White et al. 1990). Amplicons were sequenced (Macrogen, Amsterdam, The Netherlans)) and the Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/) was used for sequence similarity searches. Other fungus of Buxus colchica, were isolated by plating infected leaves spots on the surface of potato-dextrose agar (PDS) with streptomycin and 2% Malt-agar (MAS) with streptomycin in a Petri dish. After 7 days growth on MAS and PDS at 24°C obtained isolates were transferred onto PDA and MEA in Petri dish (Henricot and Culham, 2002; Šafránková et al. 2013).

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4. RESULTS AND DISCUSSION 4.1 Mtirala National Park

The massive decline of boxwood forests was observed in Mtirala National Park and Kintrishi Protected Areas during the field trips and the infected plant material for phyto-pathological studies was collected. The strong decline processes were found along the river (Figure 10; Annex A,1).

Figure 10:. Boxwood decline along the river in Mtirala National Park

In addition, the boxwood decline was found in the natural boundary in the Mtirala NP (Figure 11, Annex A,2).

Figure 11:Boxwood decline in the natural boundary in Mtirala National Park

220 isolates of Phytophthoras (from soil of examined boxwood forests) were obtained using baiting technique (Figure 12).

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Figure 12: Isolation results of soil baiting technique from boxwood forests in Colchida lowland

Among the percentage of the total number of isolates obtained from boxwood forests soils, the carnation petals and azalea gave the best results as baits for Phytophthoras. However, some isolates (M1, K5) were isolated from necrotic lesions of rhododendron leaves. The species identification of isolates and their genetic analysis require a long time of analyzes and this work continues. The results of molecular work and morphological identification of Phytophthoras will be published as soon as work is finalized and information with the link to the article will be provided. The pictures of the morphological features of isolates and pure cultures of Phytophthoras obtained from soil samples are presented in Annex A,3. Baiting is commonly used method for detection of Phytophthora spp. in soil. The technique involves floating pieces of susceptible tissue on a soil water slurry with a high water/soil ratio (Erwin and Ribiero 1996; O’Brien Philip et. al. 2009). According to the data of O’Brien Philip et. al. (2009) the efficiency of detection can range from 0 to more than 90% and depends on the time of year at which the sample was taken. The host species from which the bait tissue is derived also influences on the efficiency of detection (Marks and Kassaby, 1974). The localised spreading of Phytophthora spp. is by (asexual) spores (sporangia) produced on the surface of infected leaves and shoots (Phytophthora ramorun A practical guide for the nursery stock and garden centre industry, 2005.). The leaf hosts such as Rhododendron and possibly ash, holm oak, sweet chestnut and Vaccinium are the important sources of inoculum for initiating and maintaining epidemics of the tree mortality. Rhododendron ponticum, which growth everywhere, is a principal environmental leaf host contributing inoculum to trees (causing lethal bark infections) though some tree species with susceptible leaves may also have a role. During field trip, the infection on the leaves Rhododendron ponticum was found (Figure 13).

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a b Figure 13: Phytophthora symptoms on the rhododendron leaves: a – Phytophthoras damages on the leaves; b – brown discoloration of leaves tissues caused by Phytophthora and other saprotrophic fungus

Moreover, holm oak (Q. ilex), ash (Fraxinus spp.) and sweet chestnut (Castanea sativa). Vaccinium and Calluna may also be sources of inoculum where they occur in association with trees. Spores are likely to be spread by rain splash, wind-driven rain, water run-off, irrigation water, contaminated soil / growing media and animals (mammals are known to spread the disease including humans). Sporangia germinate under the moist conditions to release water-motile zoospores capable of spreading the disease still further. Both these spore types can be relatively long lived under humid conditions and have been found in streams, ponds and reservoirs (Phytophthora ramorun. A practical guide for the nursery stock and garden centre industry, 2005). Thick-walled, long-lived spores (chlamydospores) allow the long-term survival of the pathogen and are primarily produced within infected plant tissue. Growing media, debris and soil on vehicles, machinery, footwear or animals can also harbour and spread the pathogen. There is no evidence of any latent infection on Rhododendron leaves. Also, the bark infections was found as large cankers with brown to black discoloured outer tissues on the lower trunk that seep or, ‘bleed’ a dark red sap (Phytophthora ramorun A practical guide for the nursery stock and garden centre industry, 2005). Typically, these cankers can occur on the lower part of the trunk. When the outer bark is removed, as we see on picture below (Figure14), the mottled areas of necrotic, dead and discoloured inner bark tissue with black zonal lines around the edge may be observed. In addition, damaged trees may become colonised by bark beetles. When cankers girdle the trunk, death of the tree occurs resulting in wilting and a rapid change in foliage colour. Another aboveground Phytophthoras symptom, which was observed on Buxus colhica in Mtirala NP, had gradually signs of poor growth of the branches and abnormally small leaves (Figure 15).

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a b c

d f e Figure 14: Phytophthoras symptoms on the main stem of Buxus colchica a, b - black spots and blooding on the outside of the bark; c, d, - dark brownish-black coloration of the vascular tissues under the bark; e - black stripes in the wood; f - affected inner tissues of wood by beetles Xyleborinus saxeseni Ratz.

Figure 15: Reducing the growth of boxwood leaves and branches

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Trametes versicolor (L.) Lloyd and Stereum subtomentosum Pouzar as wood rot fungi were found on the territory of Mtirala NP (Figure16). They usually develop on the dead branches and trunks.

a b Figure 16: Fruit bodies of fungus: a – Trametes versicolor (L.) Lloyd, b– Stereum subtomentosum Pouzar.

It was also detected that the damage of the leaves with oval spots which are gradually increased. On the surface of spots were formed the black dots (bodies sporification) (Figure 17). Calonectria pseudonaviculata was isolated directly from the sporulation on the leaves and mycelium on shoots (method described above). However, the detail information about Calonectria pseudonaviculata is not emphasized as this fungus had been already detected and its role in the decline of boxwood tree stands was described (Gorgiladze et. al. 2011) (Annex A,4 ).

b

Figure 17: Calonectria pseudonaviculata on the boxwood leaves: a, b - the fungus Calonectria pseudonaviculata on the boxwood leaves; c - isolate of Calonectria a pseudonaviculata on PDA media

In addition, Volutella box blight - Volutella buxi (teleomorph Pseudonectria rouselliana) was found after keeping damaged boxwood leaves in a moist chamber that was also isolated in culture. For storage in the refrigerator, the pure cultures were transferred onto PDA media (Figure 18).

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a b

c

Figure 18: Volutella buxi on boxwood leaves: a, b – sporification on the leaves; c- isolates of Volutella buxi on PDA media

4.2. Kintrishi Protected Areas

In Kintrishi Protected Areas, the situation concerning damage of Buxus colchica is more or less similar to situation with boxwood in Mtirala National Park. Approximately 90% of all box trees lost their leaves, leaf spots and discoloration of stems have been observed and this process is still continuing (Figure 19; Annex B,1). Nevertheless, in Buxus colchica grove, the percentage of damaged and almost dead tress was extremely high (Figure 20). All trees along the river were defoliated but on several trees the signs of the native forest regeneration on the main stems have been discovered (Figure 21). The poor growth of branches and twigs were observed in Mtirala National Park as well (Figure 22).

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Figure 19: Decline of Buxus colchica Figure 20: Damaged boxwood tree in Kintrishi Protected Areas stands in Buxus colchica grove

Figure 21: The native forest Figure 22:The poor growth of Buxus regeneration on the main stems of colchica branches and twigs boxwood trees With the loss of leaves under the boxwood canopy stand the conditions for growing the alders, blackberries and herbs are being created (Figure 23). Because of this, the natural regeneration of boxwood has no chance of survival due to high competition from fast-growing plants that leads to a fundamental change in growth conditions for boxwood. 46

Figure 23: Formation of new vegetation succession under canopy of dead boxwood trees

In addition, the damage of boxwood leaves caused by other pathogens and insects was also revealed (Figure 24). Eriococus buxi Fousc. was found on the boxwood leaves near the monastery, but the massive development of these insects was not observed (Figure 25).

Figure 24: The damage of boxwood Figure 25: Eriococus buxi Fousc. on leaves the boxwood leaves

The Phytophthoras species were isolated from soil samples using baiting method. The pictures of the morphological features of isolates and pure cultures of Phytophthoras obtained from soil samples are represented in B,2. Identification of Phytophthora on these two objects is not random. The climatic conditions of the regions and sufficient moisture contribute to the spread of these pathogens. In Mtirala National Park as well as in Kintrishi Protected Areas the examined boxwood stands grow on the banks of rivers and root systems of the plants are periodically flooded in spring and during the period of heavy rains. It is proved the possibility of spreading Phytophthoras by water in 1921 (Bewley and Buddin, 1921). In their earlier works, we can find information about discovering of P. cryptogea and P. parasitica (syn. P. nicotianae) in greenhouse irrigation water. Since that time, Phytophthoras were revealed in water have been implicated in the outbreak of disease in fruit crops (Whiteside and Oswalt, 1973; Oudemans, 1999; Yamak et al. 2002), forest trees (Kliejunas and Ko, 1976) and ornamentals (Thinggaard and Andersen, 1995; MacDonald et al. 1994; Strong et al. 1997; von Broembsen andWilson, 1998; Bush et al. 2002; Themann et al. 2002). In addition, Phytophthora ramorum wihich caused the sudden oak death (SOD) has been detected in many streams and 47

watersheds in California (Maloney et al. 2002; Tjosvold et al. 2002; Murphy et al. 2005) and Oregon (Hansen et al. 2005). The fact of distribution of Phytophthora species in rivers, streams, canals and water reservoirs is well documented and established. It is known that 20 Phytophthora spp. were detected in water mainly in the USA and Europe including the most known species like P. cactorum, P. cambivora, P. capsici, P. cinnamomi, P. citricola, P. citrophthora, P. cryptogea, P. drechsleri, P. lateralis, P. megasperma, P. nocotianae, var. nicotianae, P. ramorum, P. syringae (Fergusson and Jeffers, 1999; Orlikowski, 2006; Orlikowski et al. 1995, 2007; Themann et al. 2002). Hong and Moorman (2005) noted that Phytophthora spp. may be significant crop health destructor of many plants and water may be a primary, if not a sole, source of inoculum for Phytophthora diseases. According to the research conducted by Orlikowski et al. (2009), the isolation Phytophthora citricola from rivers, water reservoirs and nursery canals during the whole year showed that pathogen’s development not depends on available temperature. This research indicates the harmfulness of Phytophthora citricola to boxwood and thuja when the first yellowing of individual boxwood shoots was found in the middle of June on about 1.5% of plants and later in 15 weeks, disease symptoms were observed on at least 10 % of plants. Besides yellowing, on about 5 % of plants most of shoots showed dark-brown discoloration. The study carried out by Matsiakh et al. (2012) in National Park „Skoliwski Beskidy” in Ukraine shows the spreading of Phytophthora species in the rivers Stryj and Yasenytsya and in the lake situated on the territory of National Park. Detection of the pathogens was done in October and November in 2011 with using baiting technique with rhododendron leaf baits cv. Nova Zembla. Phytophthora gonapodyides, P. lacustris and Pythium litorale were detected in analyzed water sources. In the literature review, to the possible danger of the spread in natural boxwood forests an invasive species Cydalima perspectalis have been mentioned. This moth has penetrated into the territory of the Caucasus from green space of Sochi Olympic Village. During the second visit to Georgia, the damage of box tree by caterpillars of Cydalima perspectalis was identified in green areas near the office of Kintrishi Ptotected Area (Kobuleti) and administration of Mtirala National Park (village Chakvi) (Figure 26), as well as in the city of Batumi and Tikeri nursery farm (Administration of Kobuleti, Lepl Forestry Agency of Ajara). The presence of Cydalima perspectalis in green spaces of towns near the protected areas with boxwood creates the great conditions for the further spread of this dangerous pest to the forests. That is why a timely and systematic treatment on small green areas may prevent its spreading. However, in protected areas effectively counteract of the pest is very difficult.

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b Figure 26: Damaged boxwood leaves by larva Cydalima perspectalis near Administartion of Mtirala National Park: a – injured plants, b – larva. a

During the summer in 2014, almost all plant material of boxwood in the nursery Tikeri farm have been destroyed. Without protect of young plants will not be able to solve the problem of natural reestablishment of boxwood plantings.

4.3. Ajameti Managed Reserve

The old-aged Imeretian oak stands have been intensive exploited for long period that contributed to the weakening of these stands and the development of great capricorn beetle (Rudnev, 1957). Since 1976, the detailed study of the main causes of oak forests decline on the territory of Ajameti has been conducted. Georgian researchers Shavliashvili I., Chapidze F., Imnadze T. revealed the progressive disease processes and decline of the oak trees in reserve (Chikovani et al. 1990). In 2007 on the territory of Ajameti, the assessment of sanitary condition of Imeretian oak forests was done by co-workers of Vasil Gulisashvili Forestry Institute (Report on forest pathology study of Ajameti Strict Nature Reserve, 2007). After examination of the oak tree stands, they determined 12 oak pathogens and 13 insect phytophagous. The authors believe that that time there was not a significant threat of oak disease and insects-phytophagous including activities of Cerambux cerdo. However, the authors point to the periodic drought and late spring frosts that can quite dramatically weakened trees. The rout (line) research for assessment the degree of the spread of pathological processes in the Imeretian oak stands and evaluation the sanitary conditions of oak tree stands was carried out on two areas in Vertsikhe and Ajameti. The examined plots were selected on the edge and in the middle of the forests. Category and classes of the distribution of trees were evaluated using IUFRO tree classification and sanitary rules of Ukraine (Sanitary Regulations ..., 1995). The presence of various pathologies including stem rot damage and insect-ksylophagy particular a great carpicorn beetle was recorded for every tree. 49

The results of the evaluation the general condition of Imeretian oak stands present the results of the distribution provisions in the forest communities. Distribution of oak trees at heights classes is shown in Figure 27. The majority of trees (55-65%) is related to the middle of the tier and their height is 1/3-2/3 of the maximum height of forest stands.

Figure 27: Distribution of oak trees at heights classes

The distribution of trees by class vitality is given in Figure 28. In the examined stands in the middle and on the edge of forests the tree of good vitality are prevailed in Ajameti and Vertsikhe and their number is 47-60%. The total number of trees of poor vitality is higher in stands located on the edge of forests.

Figure 28: Distribution of oak trees at classes of vitality Hierarchical trend allows estimating the tree position in the forest stands, the prospects for the future and longevity (Figure 29). The trees not modifying the social position (47-68%) are dominated in old-aged Imeretian oak stands. There is a number of trees (8-35%) that may worsen their social position.

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Figure 29: Distribution of oak trees at hierarchical trend

Economic evaluation of forests stands was assessed using the following parameters: silvicultural value, the quality of bole (trunk) and the length of crown. The complex of indicators has determined the trees value the main ones are breed of forest stands, its position and condition in the forest stands (Figure 30).

Figure 30: Distribution of oak trees at silvicultural value

In the surveyed plots, the helpful concomitant trees that contribute to the growth of elite trees are dominated and the number of elite trees ranges from 10 to 25%.

Classes the quality of bole (trunk) show the commodity value trees (Figure 31). The smallest number of trees with boles of good quality (8%) is found on the Vertsikhe area in oak stands older than 250.

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Figure 31: Distribution of oak trees at the quality of bole (trunk)

There is a very significant development of stem rot that reduces the quality of the wood. The number of trees of good quality increases from 32.5% at the edge of the forest to 42.5% in the centre of the area in Ajameti research plot. An indication of Imeretian oak sustainability to the external environment is the distribution by class crown length (Figure 32).

Figure 32: Distribution of oak trees at the crown lenght

A well-developed canopy of trees increases the resistance of trees to external influences. The examined Imeretian oak stands on the site of Ajameti and on the central areas of Vartsikhe are preferably formed of well-developed crown and old-aged trees. Trees with long crowns are between 30 and 55% at the edge of the forest and 20-22% in the central part of the forest. The number of trees with moderate crowns ranges from 40 to 67,5%. The concern of decline processes of Imeretian oak forests in Ajameti managed reserve has been caused for long period. The deterioration of vegetation is influenced by various factors (Shavliashvili et al. 1980; Tavadze et al., 1986). The specific approaches proposed by Manion (1991) were used for analysing the causes connected with dieback of oak forests. According to this theory, three groups of factors that can work in parallel and in series were distinguished. It is very important to the history of populations 52

and the factors that affect the weakening stands may vary depending on specific conditions. The influence of various factors on oak forests is shown in Table 3. The group of predisposing factors which act for a long time may be the main reason of weakening forests stands and lead to gradual worsening of the health conditions these forests when the symptoms are not appear in full. Among them are the origin of the plantations and an intensive economic activities (including –cutting and grazing) (Rudnev, 1957, Report on forest pathology study of Ajameti Strict Nature Reserve, 2007). Table 3. Factors affecting the deterioration of health conditions of oak forests in Ajameti I II III

PREDISPOSING FACTORS INCITING FACTORS CONTRIBUTING FACTORS

• Origin of plantations; • Spring frosts; • Changes in the functioning • Intensive exploitation and • Trunks damage by frost (frost of parasitic systems: economic activity; crack formation) • Root rot (Armillaria spp., • The composition and • The influence of high tinder fungi etc.); structure of forest stands; temperatures during the • Stem rot; • Age of forest stands summer; • Pathogens of Phytophthora • Damage caused by foliage genus; insect-foliophagi; • Ksylo- insects and • Leaves damages by powdery kambiofahy mildew

One of the reason of the deterioration of oak forest in Ajameti is intensive recreation of the areas and movement of cattle through the forests that is why in some places a dense network of roads and trails is formed (Annex С,1). The soil compaction, root “paws” injury and bark damage are being weaken the tree and create the contributed conditions for the penetration of root rot and stem rot pathogens. Grazing in the forest including pigs leads to the damage of the roots, self-seeding and the destruction of acorns harvest (Annex C,2). Many researchers talking about problems of oak forests decline in Ajameti managed reserve pointed to a significant percentage of coppice oak trees in these forests (Rudnev, 1957; Supatashvili et al. 1967, Chikovani et al. 1990). These tree stands have a lower resistance to abiotic and biotic stress factors and more intensely may affect root rot pathogens (Yakovlev and Yakovlev, 2007). According to the research conducted by Rudnev (1957), Supatashvili et al. (1968), the forest management and different types of cutting conducted in the past contributed to the weakening of oak forests in Ajameti. This has led to the increasing of the large number of oak trees coppice origin (Figure 33). The cutting of oaks also led to the formation of derivatives stands with a predominance of hornbeam, Zelkova and others trees.

Figure 33:The development of stem root rot in oaks coppice origin 53

Also, the group of predisposing factors includes the age of tree stands, as there are many oak trees older than 150-200 years (Chikovani et al. 1990; Report on forest pathology study of Ajameti Strict Nature Reserve, 2007). The stress factors that act occasionally were involved to the group of inciting factors, but they can firstly cause an intense dieback of tree stands weakened predisposing factors. A impact of frosts can be very significant inciting factor because oak is very sensitive breed to the late spring and early autumn frosts (Stoyko, 2009). Symptoms caused by late spring frosts were found in Ajameti managed reserve (Figure 34).

Figure 34: Imeretian oak leaves damage by late spring frost

Frost cracks as well as frost injures may contribute to the development of stem rot. In the examined oak stands injuries by frost cracks ranged from 3 to 25% (Figure 35). The sun heating of trees during winter also causes specific damages when at night it cools the dark well and eventually peels off the outer tissues . There are lots of such type of damages more on the outskirts of the forest than the middle of area (Figure 36).

Figure 36: Root rot in the frost Figure 35: The number of trees with frost cracks places cracks and timbers

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The dry periods may occasionally take place in Ajameti area (Report on forest pathology study of Ajameti Strict Nature Reserve, 2007) and high temperatures in spring and summer combined with the lack of rainfall may contribute to the development of insect-foliophagi. This type of damage of the assimilation system weakens oak trees and makes them susceptible to the action of insects and pathogens ksylophagy and root rot including the species of Ganoderma genus. Georgian scientists had discovered 154 species of insect pests and 54 of them were first time determined on the territory of Georgia in oak forests (Chikovani et al. 1990). The oak tree stands have been periodically damaged and as result the mass reproduction of leaves insects (in particular winter ) and stem pests: large and small oak beetle, oak Jewel and others (Shavliashvili et al. 1980; Chikovani et al. 1990). Employees from Vasil Gulisashvili Forestry Institute in 2007 discovered 13 species of insect-foliophagi that, for their opinion, significantly affect the state of oak forests (Report on forest pathology study of Ajameti Strict Nature Reserve, 2007). The list of insect-foliophagi found in Imeretian oak stands is given in Table 4. For our opinion, the additional research is needed to find out species identification, role and importance of insect herbivores in the process of oak forests decline.

Table 4. The list of insects found in Imeretian oak stands of Ajameti managed reserve Species of insect-foliophagi Hosts Type of damage Foliophagi 1 Acrocercops brongniardella (F.) Quercus imeretina and mining of the Quercus iberica leaves 2 Dyseriocrania subpurpurella (Hw.) [= Eriocrania Quercus imeretina and mining of the subpurpurella (Hw.)] Quercus iberica leaves 3 Operophtera brumata (L.) foliage tree species eating of theleaves 4 Phyllonorycter quercifoliella (Zell.) Quercus imeretina and mining of the Quercus iberica leaves 5 Rhynchaenus pilosus (F.) [= Orchestes pilosus Quercus imeretina and mining of the (F.)] Quercus iberica leaves 6 Tischeria ekebladella (Bjerk.) Quercus imeretina and mining of the Quercus iberica leaves 7 Tischeria complanella Hb. Quercus imeretina and mining of the Quercus iberica leaves 8 Tortrix viridana L. Quercus imeretina and eating of the Quercus iberica leaves Ksyloliophagi 9 Agrilus biguttatus Fab. Quercus imeretina and routes under the Quercus iberica bark 10 Cerambyx cerdo L. [= Cerambyx cerdo Quercus imeretina and routes under the acuminatus Motsch.] Quercus iberica bark and in the wood 11 L. Quercus imeretina and routes under the Quercus iberica, maple, bark and in the foliage tree species wood 13 Hylesinus fraxini Panz. ash routes under the bark 14 Scolytus intricatus Ratz. Quercus imeretina and routes under the Quercus iberica, rarely bark foliage tree species Fruit pests 15 glandium Marsh. Quercus imeretina and acorns damage Quercus iberica 55 16 Curculio elephas Gyll. Quercus imeretina and acorns damage Quercus iberica 17 Curculio nucum L. Quercus imeretina and acorns damage Quercus iberica Root pests 18 Melolontha pectoralis Germ. foliage tree species nibble the roots of young trees Pests of young tree plants 19 Andricus foecundatrix Hart. [= A. fecundator Hart., Quercus imeretina and galas on the A. pilosus Adler.] Quercus iberica branches 20 Neuroterus numismalis Fourc. [= N. numismatis Quercus imeretina and galas on the Oliv.] Quercus iberica branches

Imeretian oak leaves can be quite often damaged by caterpillars moth Tischeria ekebladella (Figure 37), which in recent years has spread widely in oak forests and sweet chestnut plantations in Georgia (Gagoshidze et al. 2012). Dyseriocrania subpurpurella may rarely damage the leaves of oak (Figure 38).

Figure 37: Mines of leaf-mining moth Figure 38: Oak leaves damaged by on the surface of oak leaves caterpillars Dyseriocrania subpurpurella

Moreover, on the surface of oak leaves leaf galls caused by Silk button gall (Neuroterus numismalis Fourc. (= N. numismatis Maur, Cunips vesiatrix Schlecht.) were found (Figure 39). Instead forming the young shoots, the pineal galls created by gall wasp Andricus foecundatrix Hart. (= A. fecundator Hart., A. pilosus Adler., A. gemmae L.) (Figure 40) were also detected on the oak leaves. However, these and similar fytophagous can only worsen the condition of young oaks. Fruit pests and root pests affect the number of self-seeding and the state of natural regeneration of oak.

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Figure 39: Galls formed by Silk button Figure 40: The pineal galls are formed gall (Neuroterus numismalis) by gall wasp by Andricus foecundatrix

The quite intensive development of powdery mildew (Microsphaera alphitoides Griffon & Maubl [= Erysiphe alphitoides (Griffon & Maubl.) U. Braun & S. Takam.]) that affects oak leaves was observed (Figure 41). This pathogen develops on the leaves of oak trees of all ages and in some cases, it may be one of the reason of significant weakening the trees.

Figure 41: Imeretian oak leaves affected by powdery mildew

Apiognomonia errabunda (Roberge) Höhn. [= Gnomonia errabunda (Roberge) Auersw; Gnomonia quercina Kleb.] may rarely cause oak leaves damages. The weakened oaks exposed to the effect of third group of contributing factors. There are mainly biotic factors, which are the result of parasites activation. Under their influence, the weakened trees will dead. This group of factors includes pathogens root rot and stem rot, activity of insects and ksylo- kambiofagi as well as the development of genus Phytophthora species. The species composition of pathogenic fungi affecting necrosis and rot pathogens were detected in Imeretian oak forest stands and related tree species in Ajameti managed reserve are listed in Table 5. These fungus samples were collected during field trips to Ajameti.

Table 5. Pathogens species composition found in Ajameti managed reserve Substrates, The types of diseases, species of fungi Hosts type of damage 1 2 3 4 Pathogens of necrosis and witch-broom disease 1 Colpoma quercinum (Fr.) Wallr. [= Clithris Quercus imeretina and the branches, necrosis quercina (Pers.) Rehm.] Quercus iberica 57

2 Taphrina carpini (Rostr.) Johanson hornbeam trunks and branches, [= Exoascus carpini Rostrup] witch's broom 3 Vuilleminia comedens (Nees) Maire Quercus imeretina and the branches, necrosis Quercus iberica Root and stem rot pathogens 4 Amillaria mellea s.l. foliage tree species stumps, dead and rarely alive trees 5 Bjerkandera adusta (Willd.) P. Karst. foliage tree species the stumps, dead and rarely alive trees 6 (L.) Pers. Quercus imeretina and the stumps, dead trees Quercus iberica 7 Fistulina hepatica (Schaeff.) With. Quercus imeretina and an old alive trees Quercus iberica 8 Fomes fomentarius (L.) Fr. Quercus imeretina and an alive weakened trees Quercus iberica, hornbeam 9 Ganoderma lipsiense (Batsch) G.F. Atk. Quercus imeretina and an alive and dead trees, [= Ganoderma applanatum (Pers.) Pat.] Quercus iberica, stumps foliage tree species 10 Ganoderma lucidum (Curtis) P. Karst. Quercus imeretina and at the bottom of the trunk Quercus iberica near the roots 11 Ganoderma resinaceum Boud. Quercus imeretina and an alive and dead trees, Quercus iberica stumps 12 Hymenochaete rubiginosa (Dicks.) Lev. Quercus imeretina and the stumps, dead wood Quercus iberica 13 dryadeus (Pers.) Murrill Quercus imeretina and alive trees [= Pseudoinonotus dryadeus (Pers.) T. Quercus iberica, rarely Wagner & M. Fisch.] malpe 14 Inonotus hispidus (Bull.) P. Karst. Quercus imeretina and alive trees Quercus iberica, rarely malpe 15 Laetiporus sulphureus (Bull.) Murrill Quercus imeretina and alive trees Quercus iberica 16 warnieri Durieu & Mont. Quercus imeretina and the stumps, dead wood Quercus iberica 17 Phellinus robustus (P. Karst.) Bourdot & Quercus imeretina and an alive and dead trees Galzin Quercus iberica 18 Stereum hirsutum (Willd.) Pers. foliage tree species the dead trees, stumps, branches, cutting remains, on the edges of lesion on alive trees 19 Stereum gausapatum (Fr.) Fr. Quercus imeretina and the dead and alive Quercus iberica, rarely weakened trees, stumps, foliage tree species cutting remains 20 Trametes hirsuta (Wulfen) Pilát [= Coriolus foliage tree species ying and dead trees, hirsutus (Wulfen) Quél.] stumps, dry branches 21 Trametes gibbosa (Pers.) Fr. Quercus imeretina and the stumps, dead wood Quercus iberica, hornbeam and foliage tree species 22 Trametes ochracea (Pers.) Gilb. & foliage tree species the stumps, dead wood Ryvarden [= Coriolus ochraceus (Pers.) Prance] 23 Trametes versicolor (L.) Lloyd [= Coriolus foliage tree species the stumps, dead wood versicolor (L.) Quél.] 24 Trichaptum biforme (Fr.) Ryvarden Quercus imeretina and the stumps, fallen dead Quercus iberica, hornbeam trees

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Necrosis pathogens Colpoma quercinum and Vuilleminia comedens develop on the lower branches of shaded oak trees, but can cause deterioration of the young trees. The development of root rot and stem rot pathogens leads to further weakening and gradual decline of trees. These pathogens very active colonize of old oak trees. 21 species of pathogenic fungi in this group were discovered on the territory of Ajameti. Amillaria spp. It is usually inhabited on living but much weakened oaks, on stumps and dead trees. Inonotus dryadeus and Ganoderma species are rot pathogens of oak. Fistulina hepatica, Fomes fomentarius, Inonotus hispidus, Laetiporus sulphureus, Phellinus robustus parasite on the weakened but alive trees. Other species settle on dead wood, stumps and some of them may live on the edges of mechanical damage to living trees. The pictures of fruiting bodies of tinder fungi found in oak forests of Ajameti managed reserve are represent in Annex C,3. The plant material for mycological analysis was collected from natural regeneration of oak self-seedlings and soil from the root system of the plants. Visual assessment of seedlings showed that the seedling’s root had the necrotic lesions, the root system was not dense and compact, and the smallest fine roots were completely rotten. Also, partially on the main root could be observed the necrosis and lesions as well (Figures 42, 43).

a b Figure 42: Sanitary conditions of the natural regeneration of oak self-seedlings: a - in June; b – in October

a b Figure 43: Infected roots of oak self-seedling: a – in June; b – in October. 59

In addition, oak trees with aboveground symptoms of Phytophthora diseases including reduction of growth and chlorosis of leaves in the nursery were observed (Figure 44) and soil sampling was conducted from rhizosphere of infecred trees.

Figure 44: The Phytophthora symptoms on the young Imeretian oak trees

Using baiting technique 163 isolates of Phytophthora from soil of Imeretian oak forests were obtained (Figure 45). The best bait for Phytophthora in this case were carnation petals. From nursery, soil could isolated more isolates but, for our opinion, the growing plants of the same age and other species in the nursery is the most favourable for the spread of root rot. The pictures of the morphological features of isolates and pure cultures of Phytophthoras obtained from soil samples are represented in Annex C,4.

Figure 45: Isolation results of soil baiting technique from Imeretian oak forests in Colchida lowland

Isolation of Phytophthora on the territory of Ajameti actually causes concern. About oak decline in Europe had been known for a long time, as well as about decline of Imeretian oak started to talk 100 years ago. This information about the possible causes of this phenomenon is described 60

in details in the literature review. However, never Phytophthora was discovered and described in the Imeretian oak forests of Adjara. Generall, there is a lot of information about Phytophthora diseases in oak tree stands. Several studies have observed deterioration of oak fine roots in Central and Western Europe (Näveke and Meyer, 1990; Vincent, 1991; Eichhorn, 1992; Blaschke,1994; Jung, 1998; Thomas and Hartmann, 1998). Isolations from fine roots and rhizosphere soil samples revealed the widespread occurrence of several Phytophthora species including P. cactorum, P. citricola, P. cambivora, P. gonapodyides, and the new species P. quercina in 29 out of 33 stands investigated in 6 European countries (Jung et al.1996; Jung, 1998). In Northern , Galoux and Dutrecq (1990) isolated a Phytophthora species from fine roots of declining oaks, and Hansen and Delatour (1999) demonstrated a diverse Phytophthora population including P. quercina in oak forest soils. In Bavaria, a survey was made on the occurrence of soil-borne Phytophthora species and the fine root status of 217 healthy and declining mature trees of Quercus robur and Q. petraea in 35 oak stands on a range of geologically diverse sites (Jung et al. 2000a, 2000b). The results showed that depending on the site conditions at least two different complex diseases are referred to under the name ‘oak decline’. P. quercina, P cambivora and P. citricola were common on sites with a mean soil-pH (CaCl2) ≥ 3.5 and sandy-loamy to clayey soil texture and seven other Phytophthora species were infrequently isolated from rhizosphere soil. Jung et al. (2000b) concluded that Phytophthora species are strongly involved in oak decline on sandy loamy to clayey sites with a mean soil-pH (CaCl2) ≥ 3.5, and defined this widespread decline type as ‘Phytophthora-mediated oak decline’ The long duration and the wide geographical range of the current episode of oak decline suggests that large-scale and long-term environmental changes in Central and Western Europe during the last decades might have imbalanced the host-parasite relationship between oaks and the naturally occurring soil-borne Phytophthora species. One major factor certainly is an excess of anthropogenic nitrogen input into forest soils that are now becoming over-saturated with nitrogen (Nihlgard, 1985; Kreutzer, 1991; Mohr, 1994; Thomas and Kiehne, 1995). Another potential threat is the continuous increase in mean winter temperatures of 0.03K per year combined with a significant seasonal shift of 20-30% of the summer precipitation into the cold season in Central Europe and parts of Western and Northern Europe since the 1960‘s (Rapp and Schönwiese, 1995; Schönwiese et al. 1994). Environmental constraints can increase the susceptibility of a host to root pathogens, and thus indirectly promote pathogen infection. However, they can also favour a pathogen directly. There are examples for both nitrogen fertilization as well as prolonged drought periods enhancing the severity of Phytophthora diseases (Schmitthenner and Canaday, 1983; Shearer and Tippett, 1989; Brasier,1993, 1996; Erwin and Ribeiro, 1996). Furthermore, the summer climate in Central Europe shows a tendency to more frequently occurring droughts and heavy rain (Schönwiese et al. 1994; Rapp and Schönwiese, 1995). During prolonged drought, periods in summertime oaks are suffering from their degenerated fine root systems thus showing aboveground symptoms of water stress and malnutrition. During periods of heavy rain, the oaks are not sufficiently able to regenerate their fine root systems because high proportions of freshly formed fine roots are destroyed in the wet soils by exploding populations of soil-borne Phytophthoras. Such a pattern of interaction between climatic perturbations and Phytophthora fine root damage was demonstrated by Brasier (1993, 1996) for Iberian oak decline caused by P. cinnamomi, and is supported by the results of our drought stress experiment. All Phytophthora species tested were able to survive repeated drought stress, and produced significant fine root damages when soils were reflooded. P. cinnamomi and one isolate of P. quercina isolated from a site with highly fluctuating water tables and very dry conditions during summertime caused 61

even more root damage with drought stress between the floodings than under non-limited water supply, whereas another P. quercina isolate isolated from a permanently moist site produced less root damage under drought stress. This might indicate to site conditions in the P. quercina population. Phytophthora "disease" of red oak () was detected in Europe (Moreau and Moreau, 1952), decline of Q. ilex and Q. suber was observed in Spain and Portugal (Brasier and Ferraz, 1993). Rapid and sometimes extensive mortality and decline of oak, principally and Q. ilex, has occurred in parts of southern Spain and Portugal in recent decades. It was reported the isolation of the aggressive root pathogen Phytophthora cinnamomi from roots of diseased oaks or from soil at eleven out of thirteen decline foci examined. It was proposed that the introduction and spread of P. cinnamomi may be a major factor in the Iberian oak decline, interacting with drought and other site factors, and leading to stress-related attacks by insects and other fungi. By analogy, it may also be involved in the similar oak declines occurring elsewhere on the Mediterranean (Brasier and Ferraz, 1993). Presence of Phytophthora spp. in the rhizosphere of healthy looking and declining Castanea sativa and trees was investigated in different forest sites in Central Italy (Vettraino et al. 1999). Five different species of Phytophthora have been isolated from soil in chestnut stands affected by "ink disease". Among these, only P. cambivora was constantly associated with symptomatic trees and isolated from infected tissues. Surveys carried out in oak stands in Central Italy revealed the presence of a number of Phytophthora species present in the rhizosphere of both healthy looking and declining trees. Among the species isolated P. citricola, P. gonapodyides, P. cactorum, P. cinnamomi and P. cambivora were identified. Four Phytophthora species, P. citricola, P. megasperma, P. quercina and P. syringae, were isolated during a systematic survey of oak forests (Quercus cerris and Q. robur) in Tuscany (central Italy), and in the Po Valley and the Venetian Plain (northern Italy) from 22 out of 54 soil samples (Barzanti et al. 2001). In the USA, some oak species die quickly whilst others decline more slowly and may take several years to die: mature oaks could be killed in less than two years (Phytophthora ramorum, A practical guide for the nursery stock and garden centre industry, 2005). The combined action of various factors lead to the weakening and drying of Imeretian oaks. The signs of weakening trees (top drying and dreid main branches) are shown in Figure 46. Also, the distribution of trees by Category sanitary condition is represnted (Figure 47). The negative impact on forest planting is revealed more on the edge of the forest. There is a great percentage of weakened trees (III category sanitary condition) drying (IV) and dry trees (V and VI).

Figure 46: Number of trees with top Figure 47: The distribution of trees by drying and dried main branches Category of the sanitary condition 62

The presence of strongly weakened oak trees in the tree stands create the conducive conditions for development of a great capricorn beetle, which in these forests had known for nearly 100 years. Many visible flying holes of great capricorn beetle were found (Figures 48; 49).

Figure 49: The remains of imago great Figure 48: Flying holes of great capricorn beetle capricorn beetle

As it was mentioned in Chapter 2, the development of a great capricorn beetle depends on the availability of weakened but alive trees. The trees with imago flying holes is higher on the edge of the forest (Figure 50) due to a presens more weak and drying trees on the outskirts of the forest. Figure 51 shows the results of counting the number of flying holes in trees of various sanitary condition. During research, any flying holes of a great capricorn beetle on alive trees with no signs of weakening were not found (I Category sanitary condition). With the deteriorating of trees health condition the number of flying holes increases.

Figure 50. Number of trees with signs Figure 51: Imago flying holes on the of settling great carpicorn beetle trees with different categories of the health status

Meanwhile, the scientists and environmentalists pay more and more attention to the preservation of dead wood in the forest. WWF believes that the existence and condition of the dead 63

wood are a kind of practical indicator of the health and biodiversity of forests (Dudley, Vallauri, 2004). Dead wood is a natural component of healthy forest. The presence of rotten wood creates the favourable conditions for development of saprotrophic or saproxylic organisms (Nieto and Alexander, 2010) (Figures 52, 53). The presence of dead wood in forests is considered as a way to improve forest management in the direction of convergence with the natural ecosystems. Sustainable management and forest conservation cannot be maintained without a significant number of fungi, vertebrate and invertebrate organisms that are functionally linking of dead wood in forest ecosystems (Dudley, Vallauri, 2004). The protection many species of vertebrates in particular bats and bird species that nest in hollows trees is a very important task. Because the bats are sensetive species, all of them are protected by Bern Convention (1979) and Agreement on the Conservation of Bats in Europe (EUROBATs). One of the main reasons for reducing the number of extremely useful small animal is a large-scale sanitary cutting that have reduced the number of hollow trees in the forests.

Figure 52: Rotten wood as a habitat of Figure 53: The lesser stag beetle saprotrophic organisms Dorcus parallelipipedus L. – the typical inhabitant of rotten wood of hardwood tree spesies

In our opinion, the old trees and dead wood should be maintained and protected on the territory of Ajameti managed reserve on the basis of the following requirements of the WWF (Dudley, Vallauri, 2004): • identifying and protecting key sites to maintain essential reference forests for deadwood species; • developing effective strategies for deadwood management within protected areas and Natura 2000 sites; • stopping removal of veteran trees and deadwood within IUCN categories I-IV protected areas; • strengthening prevention, information and education; • using active restoration measures where needed. The old-growth oak forests play an important role in fixation and preserving the carbon in ecosystems. This is one way to reduce the amount of greenhouse gases in the atmosphere (McPherson and Sundquist, 2013). That is why, Ajameti oak forests play also an important ecological role and have to be need protected and conservated. However, many old-aged and dead oaks may be the cause of possible social conflicts with local people who consider the dead wood only as an opportunity to get wood for heating. Therefore, the presence of dead and fallen by wind trees is perceived as forest mismanagement and as a source of the spread of diseases and pests to the healthy tree stands. For our opinion, a very 64

important problem for Ajameti managed reserve is that the process of Imeretian oak natural regeneration is not satisfactory. That is a threat of function this unique forests in the future. Self- seedlings of oak die under a closed canopy of trees or shrubs damaged by root pathogens and powdery mildew. Unless perform any action, it is possible to gradually replace Imeretian oak forests on the derivative forests of hornbeam, maple or, at best way, in the forests of Iberian oak. One possible way to restore Imeretian oak stands could be forest plantations. This path involves the cultivation of the plant material in the nurseries. Examination of Imereti oak nursery was carried out in June. The acorns were planted in the nursery but the young seedlings weren’t timely used for the forest planting. Currently, the height of young oaks more than 1 m and they grow very densely. Using these trees for planting is not advisable because the young oaks form a deep root system quickly. If the nursery does not operate cutting roots timely then the digging roots will be damaged and later it worsens the condition of young trees after transplantation. In our opinion, the young oak trees on this place should not be used for digging up and planting. However, over time, it is advisable to select the worse quality oak trees and cut down them. The young new tree stands may be established from the rest of young oaks on this territory in place of the existing nursery. Actually, more promising for our opinion, is promotion of the natural regeneration of oak. The oak acorns may be sown in the gaps and under canopy of trees to create a partial forest plantation. In this case they form rod root system and are not damaged after replanting.

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5. Recommendations 5.1. Proposals for the conservation management on the territory of Kintrishi Protected Areas and Mtirala National Park

According to the results of the field and laboratory studies conducted in Mtirala National Park and Kintrishi Protected Areas in boxwood forests stands the strong development of soil phytopathogens of Phytophthora genus, which cause root rot, was observed. In combination with other pathogenic fungi (Calonectria pseudonaviculata, Volutella buxi) they had also caused the intense dieback of boxwood plants in different ages. The decline process of boxwood trees is compounded by the combined action of several species of pathogenic organisms and the climatic conditions of boxwood growth. The growth conditions of Buxus colchica are confined to the valleys and river banks where it forms small groves or growing under the canopy of the other species stand (beech, alder). Wet conditions, the water level raising in the spring after the snow melts and in the summer-autumn period during heavy rains, promote the distribution of Phytophthora pathogens. Invasive fungal spores of Calonectria pseudonaviculata, which is parasitic on the leaves of the boxwood, spreads by the airflow. The wide distribution of this pathogen within Georgia can contribute as anthropogenic factor because of boxwood twigs are still used in religious ceremonies (Palm Sunday). Due to the fact that people may use the infected boxwood twigs for the ceremonies, in this way spores of Calonectria pseudonaviculata, may be spread on the long distance and later these infected branches are is often cultivated near the church. Apart from distribution this way, fungus Calonectria pseudonaviculata can spread in natural tree stands. The treatment measures and conducting some control of pathogens development in the natural boxwood tree stands is a very difficult task. Because of using fungicides on the territories of protected areas may have a negative impact on the environment (particularly on mycorrhizae, soil invertebrates and aquatic organisms). The protected status of research objects (Kintrishi protected area - IUCN category I and Mtirala National Park - IUCN category II) also does not allows using actively fungicides. After the death of boxwood trees in protected areas, the secondary succession begins that leads to the undesirable change of plant vegetation. Moreover, the boxwood leaves death increases the supply of light under the canopy tree stands with box tree participation. As a result, herbs (particularly - blackberries) are overgrown everywhere and the alder shrubs rapidly renewable, which make renewal the box tree in the natural way difficult or practically impossible task. It is concerned about penetration in the neighboring zones of protected areas the new invasive species butterfly Cydalima perspectalis. During the second visit to Georgia (October 2014), we had identified the damage of box tree by caterpillars of this butterfly in green areas near the office of Kintrishi Protected Areas (Kobuleti) and administration of Mtirala National Park (village Chakvi), as well as in the city of Batumi and Tikeri nursery farm (Administration of Kobuleti, Lepl Forestry Agency of Ajara). Taking into account this new finding, there is a real opportunity the spread of this invasive pest in the natural box tree stands. As a one way of treatment against caterpillars of Cydalima perspectalis in green areas is using the insecticide of enteric contact action, particularly synthetic pyrethroids: Sumi-alpha (Esphenvalerat, Senpai); Decis, Decis prophi (Kotryn, Oradelt, Polytox,) Fastak (Alphatoks, Geletrin, Kinimks, Fury) and others. However, the most of these synthetic preparations do not give the proper effect at high temperatures of air and they are toxic to bees and are banned for use near the water. 66

It is not advisable to apply these synthetic pyrethroids in the protected areas. In any case, all synthetic preparations used for pest treatment have to be registered and approved for apply on the territory of Georgia. The preparation Dimilin may be used for destroying the caterpillars of Cydalima perspectalis in the forest plantations, which is an inhibitor of chitin and has no effect on other organisms, except insects. This chemical has been well established against different species of filophagi insects. Other invertebrates (as water and land) are resistant or weakly resistant to "Dimilin." As well as, it refers to a low-hazard insecticides, has a low toxicity to mammals, birds and fish. However, to determine the feasibility and efficiency of use this preparation against caterpillars of Cydalima perspectalis is needed to conduct several additional tests. However, it would be the optimal solution to use the bacterial preparations made based on the bacterium Bacillus thuringiensis, in particular Bitoksibatsillin. Thus, in our opinion, on the territory of studied areas, the dieback of the existing boxwood trees has already reached an irreversible process stage. The studied old growth boxwood trees in Kintrishi Protected Areas and Mtirala National Park are almost completely dead. The restoration of box trees in these areas is only possible with the active and long-term human intervention and well thought out forest management. The artificial boxwood trees restoration is only possible with combination measures to prevent the escalation of blackberry, alder and other shrubs. The fragments of younger boxwood bushes preserved along the river banks, in particular near the shelter for visitors (Kintrishi Protected Areas). In such areas, the decline of box tree processes had been found also, but there are still growing not damaged plants. In order to restore box tree stands the following tasks may be recommend: • In dieback areas of boxwood tree stands it is needed to distinguish and to take samples of resistant plants and use them for the cultivation of seedlings in the nurseries for further artificial regeneration of boxwood plantings. • After determining the pathogen species composition of Phytophthora genus it is needed to perform the special work to find species-antagonists (, fungi) that can be used to increase the biological sustainability of boxwood tree. • It is necessary to carry out the inoculation tests on boxwood tree seedlings for testing the biological preparations, which can be used against the Phytophthora diseases. • The penetration of new invasive pets of Cydalima perspectalis into boxwood plantings requires protective action against this species. For this purpose, the rangers of protected areas should monitor the emergence of this pest into the natural bow tree stands. In addition, the further studies should determine the treatment preparations that can be used in the settlements (in green areas), forests and on the territories of protected areas against the damaging of box tree leaves by caterpillar of Cydalima perspectalis.

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5. 2. Proposals for the conservation management on the territory of the Ajameti managed reserve

For our opinion, the conservation activities on the territory of the Ajameti reserve should be developed according to the following three directions: • Protection and conservation of the best-preserved areas of old-growth Imeretian oak stands. • Restoration of forests stands with a predominance of Imeretian oak in trees composition. • Activities in the field of environmental education, tourism and recreation. During conducting the forest management planning activities, it should be developed a scheme of functional zoning of the reserve and differentiated regalement for conservation activities of its various parts. It is possible to select such areas: • The zone of special protection. It should include the best-preserved old-growth Imeretian oak stands. It is not necessary to perform any economic activities to avoid disturbances in natural processes. • The areas where it is needed to carry out forestry activities for support of Imeretian oak natural regeneration and maintenance of existing self-seeding. Such activities should be conducted to prevent of overgrowth grasses (in particular blackberries) in the gaps inside tree stand and to thin out (cut) self-seeding shrubs and secondary tree species in order to ensure the better conditions for growth and development of Imeretian oak self-seeding. If self-seeding process is characterized by insufficient quantities, it is also necessary to carry out the sowing of Imeretian oak acorns. • The area of derivative stand. In the derived stands with a predominance of hornbeam or Zelkova the measures of reconstruction and restoration of Imeretian oak forests оn such areas should be conducted. • The strongly affected areas with paths, roads and with trampled grass large places. The measures to restore of Imeretian oak stands on these territories will require long-term efforts and considerable resources. While creating the under canopy plated stand it is necessary to fence the young trees and prevent their damage or trampling by wild animals and livestock.

Ajameti managed reserve consists from the three clusters of different sizes. The Ajameti station is the most promising for restoring of Imeretian oak is. The smaller clusters – such as Vertsikhe and Sviri - require a different forest management regime. The forest management must correspond to the state of Imeretian oak stands in different parts of the reserve in order to be able to decide whether the selection of certain areas and the possible mode of economic intervention are reasonable. Proposals for the environmental activities in Ajameti reserve are presented below.

5.2.1. Protection and conservation the best-preserved areas of old-growth Imeretian oak stand

On the territory of Ajameti managed reserve there is a conflict of interests between the two species of the Red List of IUCN, which should be protected, Imeretian oak and great capricorn beetle (Cerambyx cerdo). The great capricorn beetle (Cerambyx cerdo, Linnaeus, 1758) together with other the major xylophages and sarcophagus, in particular with stag beetle (Lucanus cervus Linnaeus, 1758) and hermit beetle (Osmoderma sp.), is a kind of umbrella species that represents the diversity of the 68

endangered fauna associated with old oaks (Ranius, 2005; Buse et al., 2008; Ducasse and Brustel, 2008; Chiari et al., 2012). Cerambyx cerdo disappeared in the UK (Alexander, 2002), its population is rapidly reducing in the northern part of the areal (Sláma, 1998; Ehnström and Axelsson, 2002; Starzyk, 2004; Jurc et al., 2008; Ellwanger, 2009), but it is still relatively high on the south, in particular on the Caucasus (Sláma, 1998; Sama, 2002; Miroshnikov, 2009). The centres of Cerambyx cerdo mass reproduction had been observed in different years on the Caucasus: in Georgia and Abkhazia (Lozovoy, 1956; Rudnev, 1957), in Armenia (Lozovoy, 1941; Mirzoyan, 1977), Azerbaijan (Samedov, 1963), in the North Caucasus (Dobrovolsky, 1951; Miroshnikov, 2009). Miroshnikov points out that on the Black Sea coast in recent decades the Cerambyx cerdo population remains high although it occurs on the small areas or on the individual trees (Miroshnikov, 2009). The decrease of great capricorn beetle habitats under the influence of cutting mature forests and as a result of reducing the number of separately growing old oaks caused a decrease the number of this species within its global population (Nieto and Alexander, 2010). Currently Cerambyx cerdo is included in the IUCN Red List criteria categorized as Vulnerable VU B1a + B2a. The economic impact on tree stands in Ajameti reserve and the cuttings in the past caused significant number of trees coppicing, which strongly affected by root rot. This is also one of the reasons of long history the great capricorn beetle existence in oak forests in Ajameti where weakened but live oak trees are still feeding grounds of this xylophagy. For our opinion, the active measures against great capricorn beetle in Ajameti, are not needed. Greater attention should be focused on reproduction of Imeretian oak stands by promoting its natural regeneration or, if necessary, planting oak forests. For the derived plantings that had emerged because of forest management in previous decades it is worth to provide reconstruction of these tree stands. On the territory of Ajameti managed reserve the process of falling away old-growth trees is ongoing (due to the presence of stem rot, damages by wind, they breaking down). . As a result, there are two possible scenarios. • The natural regeneration of Imeretian oak in the areas where it is fallen down by wind can lead to the gradual formation of different ages tree stands. For intensification of the process of natural regeneration in the conditions of the nature reserves the creation of under canopy stands and partial planting cultures by sowing Imeretian oak acorns (shpyhovky) should be performed. • The growth of secondary tree species and shrubs (including - rhododendron, cornus ets.) in the gaps which are being arisen. In this case undesirable tree species change will take place with the formation of derived tree stands with a predominance of hornbeam, Zelkova and maples in tree stands compositions.

Moreover, the work of cutting down and removal of old decline trees, dead wood, wind fallen trees should be limited because such elements are associated the rare species of mosses, lichens and insects-xylophagy (including great capricorn beetle and other species). During the forest management operations it is necessary to distinguish areas where processes of functioning the old growth oak stands (like natural forests) are the best occurred. These places can be a kind of "cores of biodiversity" and should be placed in the middle of the forest, away from roads and livestock routes places. Moreover, there should be limited any activities except perhaps only promotion of Imeretian oak natural regeneration. The measures of sowing acorns, litter loosening, and prevention of overgrowth blackberry should be primarily done in the 69

windows are formed by fallen trees and windfalls. The fallen trees should be left for rotting and destruction by insects-xylophagy. Later, on the dead-wood and weakened trees xylophagy fungi, insects and xylophagy sarcophagus will grow (including great capricorn beetle). The protection and conservation the old-growth forests including damaged by stem rot or dead trees currently is considered as an important task for foresters and environmentalists (Dudley and Vallauri, 2004). To clarify the recommendations of the management of these "cores of biodiversity" some research plots should be studied where it is needed to elaborate methods and techniques to support natural regeneration and provide monitoring of the processes that take place there.

5.2.2. Restoration of forests with a predominance of Imeretian oak

The processes of natural regeneration in Ajameti reserve are complicated because of several reasons: • A close canopy of old trees under which acorns grow may prevent the development of self- seeding because of the absence of light. It is the reason that by the end of summer or the following year the young oak trees are affected by powdery mildew and often die; • Root pathogens (Phytophthora genus) negatively effect on self-seeding and undergrowth of Imeretian oak which can also be a threat to oak stands, especially after its weakening as a result of abiotic factors (dry years, changing rain periods) or under the influence of biotic factors (leaf damage by insects- foliophagus); • Development of forests gaps under canopy of maternal tree stand naturally well renewed related tree species (hornbeam, oriental hornbeam, Zelkova), grasses and shrubs, which quickly close up and worsen the growth conditions and development of self-seeding of Imeretian oak.

Promotion of Imeretian oak natural regeneration in the forest tree stands with a predominance of this species should include: 9 Loosening of litter in the yields years; 9 Sowing acorns in the areas of insufficient oak natural regeneration; 9 Preventing blackberry and other shrubs overgrowth in the forest gaps.

The reconstructive measures of derived plantations should be conducted with a view to reproduce the native forest tree stands. Wood, harvested in these areas, may be sold to local population and it will cover partially the costs of reforestation. During the logging, Imeretian oak trees should be left. In addition, the reconstruction of derived tree stands should be carried out with forming windows firstly cutting down the gaps with a diameter multiple 1-1.5 height tree stand. 2-3 years before starting work in these windows is necessary to sowing acorns or planting previous under canopy planted areas. It is needed to provide treatments of oak young trees in the formed gaps. If it will be necessary, these areas should be fenced in order to prevent young trees damage by animals. The acorns sowing or "shpyhovka" is one of the possible methods how to reproduce Imeretian oak. Acorns should be sown in rows (distance between rows 3-4 m; 0.25-0.3 m in a row). Moreover, the acorns may be sown as the groups in order to form biogroups of young Imeretian oak trees under canopy of old-growth tree stands. 70

Method "shpyhovka" (Kolesnychenko, 1981) includes next steps: using a shovel (or hoe) will make a sloping soil rift depth of 6-8 cm and without removing the blade, putting two acorns in it (Figure 54). Then, a shovel should be pulled out; sloping layer of the ground under its own weight will fall and close the rift and then it may be pressed by foot in order to improve the contact of acorns with the ground.

Figure 54: Shpyhovka (sowing) acorns under the blade (by Kolesnychenko, 1981

Using of oak seedlings for the regeneration of oak forests gives it worse results. During the growing of oak planting material in the nurseries, the main root is often cut in order to form the fibrous root system. If it would be not made, when digging seedlings the main root will be broken off and the young tree after replanting long time will remain weakened without getting the essential nutrients for its development. Although the fibrous root system of oak forest plantations forms satisfactorily, in the future they may be weakened and dieback. Oak is a tree which in natural conditions creates a deep root system that allows it to withstand an extreme dry periods due to the penetration of roots in the deeper soil horizons. Even with a well-formed in the nursery fibrous root system, the bulk of the oak roots will be focused in the upper soil horizons making these spaces more susceptible to drought. Moisture deficit, in turn, may provoke stronger damage of the assimilation system by leaves-feed insects and will contribute to colonization by necrosis and cancers pathogens and vascular diseases that can lead to weakening and dieback of oak forests. For future conversation and restoration of Imeretian oak strict control and monitoring visits to Ajameti reserve should take place. In addition, it is important to prohibit the cattle grazing in these forest stands.

5.2.3. Environmental, educational and recreational activities

Also, the attitude of local population is a problem in the Ajameti managed reserve. The demand of wood for heating causes dissatisfaction especially when damaged trees and deadwood are not cut down and not used at all. Therefore, there is a need for environmental education activities to raise public community (schools, young people and adults) to implement the protection and restoration measures of Imeretian oak tree stands. For this purpose, it is necessary to prepare a series of the science popular publications with the explanations: • a role and a significance of forests which are dominated with Imeretian oak; 71

• a role and a significance of ksylo- and saprotrofagus organisms, their functions in ecosystems; • an impact of economic activities (grazing, grass burning, agricultural activities, recreation) on Imeretian oak forests.

In addition, the interests of the community should be taken into account. For this purpose, it should create several environmental and educational paths, recreational places and to equip them with small architectural forms (benches, canopies, sold-out information etc.). Conducting works on the reconstruction the low value derivatives tree stands will allow the use of wood by the local population. In the future, the attention should be focused on the development of organized tourism in Ajameti managed reserve. The main idea there may be as "We won’t lose what the Georgian kings saved." Under this slogan, the weekend trips on the reserve’s territory may be offered with the interesting tourist routes for organized groups of visitors and tourists. Ajameti managed reserve may be one of the nice components of the adventure tours by car on the way to mountain passes Sairme and Zekari in Abastumani Region. However, for developing the tourism it would be necessary to create the main customer service in Ajameti managed reserve that would include the network with rest houses, shelter, mini- hotels, recreation centres, cafes, bars and so on. It is also possible to form especial conditions for sport and providing medical care for tourists and reserve visitors. This network may be created by the Administration or Reserve management office with involving the local people by providing permits for business activities.

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Other Findings and Research

The bleeding lesions on the alder’s main stems were found on the territory of Mtirala National Park (Figures 55).

Figure 55: Phytophthora symptoms on the alders main stems

These lesions are typical symptoms of Phytophthora diseases. Moreover, the alders are growing with boxwood trees on the banks of river. The isolation of Phytophthora directly from the infected tissues wood are specific since alder bark contains polyphenols that inhibit the growth of fungi. In order to remove excessive polyphenols and stimulate germination of resting spores and thus increase the isolation frequency, the bark samples of alders and beech trees have to be regularly flooded and washed for a couple of days before plating (Jung and Blaschke, 2004; Cech, 2004; Gibbs et. al. 1999). Typical Phytophthora symptoms were also found on the stems of eastern hornbeam Carpinus orientalis in Ajameti managed reserve (Figure 56).

Figure 56: Phytophthora symptoms on Carpinus orientalis on the main stems

As it has already been mentioned, Rhododendron spp infected by Phytophthora species deserve special attention in these studies. Leaf hosts such as Rhododendron and possibly ash, holm oak, sweet chestnut and Vaccinium are therefore important sources of inoculum for initiating and maintaining epidemics of tree mortality. Rhododendron ponticum, which grow everywhere, is a 73

principal environmental leaf host that contributes inoculum to trees (causing lethal bark infections) though some tree species with susceptible leaves may also play some role. Phytophthora symptoms were also observed on several trees of Juglans spp. (Figure 57).

Figure 57: Phytophthora symptoms on Juglans spp. main stems

For our opinion, the presence of Phytophthora diseases on Juglans spp. may be a problem for cultivated fruit trees in the gardens and plantations. This work requires further detailed studies. During the field trips, the attention was also paid to a massive and intensive decline of sweet chestnut in Mtirala National Park and Kintrishi Protected Area (Figure 58).

Figure 58: Decline of chestnut in Mtirala National Park

The fungal pathogens and saprobes of chestnut (Castanea sativa Mill.) have been widely discussed during the last few years. The fungi Cryphonectria parasitica (Murr.) Barr, Phytophtora cambivora (Petri) Buism. and Ph. cinnamomi (Rands.) have been recorded as the most dangerous agents in Europe (Robin and Heiniger, 2001; Vannini and Vettraino, 2001). Some fungal species namely, Melanconis modonia (Tul.) Hohn, Cryphonectria radicalis Fries and Mycosphaerella maculiformis (Pars.) Scherut are also known to cause damage to chestnut (Diamandis, 2001). Cylindrosporium castaneae (Lev.) Krenner, Cytospora intermedia Sacc., Pseudovalsa madonia (Tul.) Hohn., Coryneum modonium (Sacc.) Crifon et Maubl, and Phomopsis castaneae Woronich have also been frequently found on sweet chestnut (Juhásová, 1999). 74

Ink disease is one of the most destructive diseases affecting Castanea sativa Mill. It causes root and collar rot of adult trees and of seedlings in nurseries, plantations and forests (Vannini and Vettraino, 2001). Symptoms of the disease on adult trees include chlorotic leaves reduced in size, thinning of the crown, and immature husks remaining on the tree after leaf-fall. Flame shaped dark necroses are evident on the collar of the tree after debarking. It is the large roots that are mainly infected. They produce a black exudate that stains the surrounding soil, especially during spring and fall. On young trees with smooth bark, the necroses are visible without debarking as depressed, slightly cracked areas at the base of the stem. Infected seedlings in nurseries or plantations undergo a rapid or gradual wilting of the leaves. In the root system, there is extensive necrosis of the tap root that extends to the lateral roots and up the stem for some centimetres. Phytophthora cambivora and P. cinnamomi are the two species most commonly associated with the disease in Europe and considered the most pathogenic to C. sativa. These species are able to kill adult trees in one to three years. P. cactorum and P. citricola have been recently isolated from soils in chestnut stands affected by ink disease. As was described in Literature Review, Venediyapina (1985а) mentioned for the first time about Phytophthora disease in Abkhazia in 1985. It was the discovery of Phytophthora cinnamomi in soils of chestnut woods Abkhazia. In addition, Tavadze B. studied the main disease of Castanea sativa in Georgia (Tavadze, 1982). During the field trip in June-July, the affected chestnut shoots with necrosis on the branches were collected for further laboratory analysis (Figure 59).

Figure 59: Symptoms of Cryphonectria parasitica on the affected chestnut shoots

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Fungal isolations were made by transferring the infected plant tissue to MEAS (20g malt extract, 20g agar per L water) with streptomycin after it was sterilised several time in alcohol and distil water. Pure cultures were transferred onto PDA media for storage. The fungi Cryphonectria parasitica (Murr.) was isolated from the infected branches of Castanea sativa (Figure 60).

Figure 60: Isolates of Cryphonectria parasitica obtained from affected chestnut shoots

Phytophthora symptoms were observed on the leaves of chestnut in Mtirala National Park and Kintrishi Protected Area (Figure 61).

Figure 61: Phytophthora symptoms on the sweet chestnut leaves

For our opinion, the study of the chestnut decline phenomenon in forests tree stands in Georgia requires further deeper phytopathological and genetic researches and should be continued in this scientific direction. The damage of the chestnut leaves caused by caterpillars Tischeria ekebladella (Bjerkander) was found in Mtirala NP. According of the observations made by ranger of Mtirala NP, a very strong development of this pest stated during the last years and may be a serious weakening factor for sweet chestnut (Figure 62).

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Figure 62: Tischeria ekebladella (Bjerkander). on the leaves chestnut

A very rare and interesting lichen Lobaria pulmonaria (L.) Hoffm. (family Lobariaceae) was discovered on the territory of Mtirala National Park and Ajameti managed reserve (Figure 63). For our opinion, more attention should be paid to this lichen in future studies of these protected areas. Lobaria pulmonaria is a large epiphytic lichen consisting of an ascomycete fungus and a green algal partner living together in asymbiotic relationship with a cyanobacterium—a symbiosis involving members of the three kingdoms of organisms. Generely known by various names like tree lungwort, lung lichen, lung moss, lungwort lichen, oak lungs or oak lungwort, it is sensitive to air pollution and is also negatively affected by habitat loss and changes in forestry practice.

a b c Figure 63: Lobaria pulmonaria in Mtirala National Park: a – the thallus of Lobaria pulmonaria, b - leathery and lobed with a pattern ridges and depressions on the upper surface, c - the thallus of Lobaria pulmonaria on the bark of Imeretian oak in Ajameti managed reserve

Its population has declined across Europe and L. pulmonaria is considered to be endangered in many lowland areas. The species weseused in herbal medicines in the past and recent research has corroborated some medical properties of lichen extracts. It is a foliose lichen and its leaf-like thallus is green, leathery and lobed with a pattern of ridges and depressions on the upper surface. Bright green under moist conditions, it becomes brownish and papery when it is dry. This species often have a fine layers of hairs and a tomentum 77

on its lower surface. The cortex, the outer protective layer on the thallus surface, is roughly comparable to the epidermis of a green plant. The thallus is typically 5–15 centimetres (2.0–5.9 in) in diameter, with individual lobes 1–3 centimetres (0.39–1.18 in) wide and up to 7 cm long. The asexual reproductive structures soredia and isidia are present on the thallus surface. Minute (0.5– 1.5 mm in diameter) cephalodia—pockets of cyanobacteria—are often present on the lower surface of the thallus; these spots are conspicuously darker than the green surface of the thallus. Like other foliose lichens, the thallus is only loosely attached to the surface on which it grows. Lobaria pulmonaria has a very significant area of distribution: Europe, the Caucasus, the Urals, Siberia, the Far East, North America, Africa. It is spread in boreal, temperate, mountain and ocean areas (Yoshimura, 1998), where it grows on the bark of various trees (coniferous and deciduous) in different types of forests. However, the vast majority of the territory of the range occurs singly or in small groups. In the boreal forest of northern Europe, it occurs in the forests of Norway spruce (Hakulinen, 1964; Istomina, 1996; Kuusinen, 1996), in hemiboreal and temperate zones of Europe - more common in deciduous forests. It can grow under the conditions of different lighting, but it is considered that the species are high-light susceptible (Gauslaa and Solhaug, 1999) and require high air humidity (Pannewitz et al. 2003). L. pulmonaria as medicine is still used for asthma, urinary incontinence and lack of appetite. In India it is used as a traditional medicine to treat hemorrhages and eczema, and it is used as a remedy for coughing up blood by the Hesquiaht in British Columbia, Canada. An ethnophytotherapeutical survey of the high Molise region in central-southern Italy revealed that L. pulmonaria is used as an antiseptic, and is rubbed on wounds. L. pulmonaria has also been used to produce an orange dye for wool, in the tanning of leather, in the manufacture of perfumes and as an ingredient in brewing. In the last century in Europe the population of Lobaria pulmonarya was sharply reduced. The lichen is very sensitive to air pollution especially for sulfur dioxide and acid rain. These factors and forestry activities limit its spread, including violation of habitat as well as a result of logging (James et al. 1977; Wolseley and James, 2000). Anthropogenic changes in forest ecosystems complicate the distribution of this species (Werth et al. 2006a) and influence on the opportunity of the colonization of new substrates determined by the level bark pH (Wolseley and James, 2000) and the degree of coverage of host trees (Öckinger et al. 2005). This was the reason that L. pulmonaria was included in the national Red Data Book in many European countries (Randlane et al. 2008; Jüriado and Liira, 2009). However, in the Caucasus conditions for the existence of a population of this lichen are more suitable (Urbanavichus and Urbanavichene, 2012). Even though, it is believed that Lobaria pulmonaria is clearly visible as indicator, which is easy to detect and identify even for workers or forest rangers. The detection of L. pulmonaria in specific areas suggests that in these areas other lichens may be distributed, which experts- lichenologists can only identify.

On the branches and stems of Buxus colchica on the territory of Mtirala NP and Kintrisi PA an intensive growth of moss was revealed. It is considered that this phenomenon may be the cause of decline of boxwood. Currently, the bryologists of the Institute of of NAS of Ukraine have determined several species of mosses. In particular, the moss Hypnum cupressiforme Hedw (cypress-leaved plait-moss or hypnum moss) is often spread and widely distributed in different climatic zones on all continents except of Antarctica. This moss grows on the trunks of trees, logs, walls, buildings, rocks and other surfaces. It prefers acidic environment and is fairly tolerant to pollution. 78

The moss Neckera crispa Hedw.is often used as an indicator of old-growth deciduous forest class Querco-Fagetea and was also discovered in Mtirala NP. According to the information of specialist’s bryologists, the mosses can not be the cause of deterioration and decline of boxwood forests. They are indicators of the wet habitats on the areas where the optimum temperature conditions are favourable for the development of pathogenic fungi. For our opinion, on the territory of examined protected areas it is necessary to conduct specialized lichenology and bryology research during which it will be possible to discover rare lichens and mosses. The results of this survey can be the basis for monitoring the environment of these protected areas.

During the second field trip in October with aim to check sanitary condition of the planting material of Buxus colchica we visited the Tikeri nursery farm (Administration of Kobuleti, Lepl Forestry Agency of Ajara). The intensive decline of the young plants strongly damaged by caterpillars of Cydalima perspectalis was observed and after chemical treatment in 2014, only the individual young boxwood seedlings remained. The plantation of fast-growing poplars is established on the territory of this nursery. The damages with characteristic necrotic lesions and cancers caused by the fungus Cytospora chrysosperma were revealed on the infected branches of poplar (Figures 63, 64).

Figure 64: Brown pycnidia on the Figure 65: Cancer lesion on the trunk surface of necrosis lesion of a poplar

Aspen green sawfly larvae Lygaeonematus compressicornis F. (Figure 65) was found on the poplar leaves and it may affect the different species of poplar. The individual leaves were damaged by poplar rust fungi of the genus Melampsora (Figure 66) and various herbaceous plants, pine or larch needles can be as an intermediate hosts of these fungi.

79

Figure 66: The larva of aspen green Figure 67: Rust on the surface of the sawfly poplarleaves

The recommendations of treatment measures against identified pests and pathogens were sent to the Administration of Kobuleti, Lepl Forestry Agency of Ajara. However, all control measures on this poplar plantation must agree with the company that created this plantation and is responsible for the preservation healthy plant material.

80

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96 Annex A Mtirala National Park

1. Boxwood decline along the river in Mtirala National Park

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2. Boxwood decline in the natural boundary in Mtirala National Park

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Isolate M1 from Buxus colchica Isolate M4 from Buxus colchica

Other Phytophthora isolates Isolate M5 from Buxus colchica 3. Morphological features and pure cultues of Phytophthora isolates obtained from soil samples in Mtirala NP

a b c

4. The damage on boxwood leaves: a – affected leaves; b – sporification on the branches after being in a moist chamber; c – isolate of Calonectria pseudonaviculata on PDA media

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Annex B Kintrishi Protected Area

1. Damaged boxwood tree stands in Buxus colchica grove

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Isolate K1 from Buxus colchica Isolate K3 from Buxus colchica

Isolate K4 from Buxus colchica Isolate K5 from Buxus colchica

Isolate K7 from Buxus colchica 2. Morphological features and pure cultues of Phytophthora isolates obtained from soil samples in Kintrish PA

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Annex C Ajameti Managed Reserve

1. Paths and roads - the consequences of the recreational using the areas and pasturing livestock

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2. Grazing including pigs in the oak forests

Trametes gibbosa Lenzites warnieri

Trichaptum biforme

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Stereum gausapatum Stereum hirsutum

Fomes fomentarius Hymenochaete rubiginosa

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Trametes ochracea Trametes hirsuta

Phellinus robustus Daedalea quercina

Ganoderma lucidum Ganoderma lipsiense [= Ganoderma applanatum]

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Ganoderma resinaceum

Fistulina hepatica Inonotus hispidus

Laetiporus sulphureus Armillaria spp. 3. Fruit body of fungi found in Ajameti managed reserve

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Isolate A1 from Quercus imeretina Isolate A2 from Quercus imeretina

Isolate A3 from Quercus imeretina Isolate A4 from Quercus imeretina

Isolate A5 from Quercus imeretina Isolate A7 from Quercus imeretina

Isolate A8 from Quercus imeretina Isolate AN1 from Quercus imeretina 4. Morphological features and pure cultues of Phytophthora isolates obtained from soil samples in Ajameti managed reserve

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Last page

Firstly, I would like to thank for an opportunity to do this research as an international consultant of the European Neighborhood and Partnership Instrument East Countries Forest Law Enforcement and Governance II Program. Whith the help of this work I discovered an incredibly beautiful country where you want to go back and enjoy the scenery and landscapes. These vivid memories of my trips to Protected Areas will forever be in my memory as one of the warmest and unforgettable period in my life. I want to thank everyone who assisted me to do this work and for the help to organize my field trips and everyone who contributed to solving all working tasks. Among them: Davit Khomeriki Director of Mtirala National Park Administration Amiran Khinikadze Director of Kintrishi Protected Areas Administration Gocha Ghibradze Director of Ajameti Managed Reserve Administration

The help of forest rangers of these research areas was very useful. I would like to thank Ruslan from Mtirala National Park for his love of the forest and for his help in collecting plant material and for the delicious mergelski khachapuri with eggplant and . I also want to thank Merab Gugitidze (Kintrishi Protected Areas) and Artem Giorgadze, Merab Zivzivadze, Avtodil Arshilava, Temur Kakhiani (Ajameti Managed Reserve) who accompanied me on my field trips to the forest and are genuinely concerned about the heath conditions of Buxux and Imeretian oak on their territories. Special thanks to Merab Machavariani who helped me with the field trips and performed this task perfectly. I am very also grateful to Administration of Agency of Protected Areas in Georgia, namely Khatuna Tsiklauri (Main Specialist) for her assistance in solving work issues and for the warmth and sincerity that I met there. I am grateful to Marika Kavtarishvili ENPI FLEG Country Program Coordinator of Georgia (Acting Head of Office, Caucasus Cooperation Center) for the huge office work and regular comminication during the last of half year. Since the beginning of our cooperation, she has been helping and supporting me all the time. I hope that we will keep in touch and friendships will last after this project finishes. One more thank Dr. Anna Maria Vettraino for her agreement to take me in the DIBAF University of Tuscia, to do some laboratory research and to share her experience and knowledge with me. I really appreciate her love and friendship. This experience would not have happened without care and love of my mother and daughter. Finally, the belief in me and continuous support of my boss and friend V. Kramarets has allowed me complete this research and improved my scientific experience.

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P.S. Unforgateble memories in pictures…

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 About FLEG II (ENPI East) Program

The Forest Law Enforcement and Governance (FFLEG) II European Neighbourhood annd Partnership Instrument (ENPI) East Countries Program supports participating countries’ forest governance. AAt the regional level, the Program aims to implement the 2005 St. Petersburg FLEG Ministerial Declaration a nd support countries to commit to a time-bound action plan; at the nationaal level the Program will review or rev ise forest sector policies and legal and administrative structures; and improve knowledge of and supporrt for sustainable forest management and good forest governance in the participating countries, and at the sub -national (local) level the Program will test and demonstrate best practices for sustainable forest manageme nt and the feasibility of improved forest governance practices at the field-level on a pilot basis. Participating coountries include Armenia, Azerbaijan, Belarus, Georgia, Moldova, Russia, and Ukraine. The Program is funded by tthe European Union. http://www.enpi-fleg.org  Project Partner

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