The Most Important Parasitic and Saprophytic Fungi on Flowering Ash ( Fraxinus ornus ) in Parks of serbia and Montenegro

In order to fulfil the gap in domestic literature about biotic causes of flowering ash ( Fraxinus ornus ) decline in urban conditions, this paper presents the results of a five-year investigation of the most important parasitic and saprophytic fungi on this tree species in parks of Serbia and Montenegro. In total, 21 fungal taxa were recorded. Within recorded taxa, 2 taxa were found on leaves, 1 taxon was found on root, thin branches and bark, while 16 taxa were found on the trunk. On leaves Ascochyta spp . was recorded. The most significant fungi were Armillaria mellea , Phellinus igniarius aff. and Inonotus hispidus . Species Meripilus giganteus and Schizophyllum commune were the main successive fungi and were often found on substrate damaged by the most significant fungi or abiotic disorders. Species Hymenoscyphus fraxineus was recorded on single trees, as well as taxon Neonectria spp. The majority of recorded taxa, including invasive pathogen Hymenoscyphus fraxineus were for the first time found on flowering ash ( Fraxinus ornus ) in Serbia and Montenegro, especially in urban conditions of these countries. Better protection strategies of flowering ash ( Fraxinus ornus ) in this part of the region could be achieved based on these results.

Genus ash (Fraxinus) contains about 60 species of broadleaved trees and shrubs and it is divided into 7 subsections (Cvjetićanin et al. 2016). Flowering ash (Fraxinus ornus) belongs to subsection Ornus and represents its typical species with flowers terminally or laterally in paniculate inflorescences on young twigs (Cvjetićanin et al. 2016). The species is autochthonous and a heliophyte; it belongs to sub-Mediterranean flora, grows in thermophilic oak forests, thermophilic bushes and occurs on dry, shallow soils (Cvjetićanin et al. 2016(Cvjetićanin et al. , Šeho et al. 2019. Also, flowering ash (Fraxinus ornus) is a biomeliorative and decorative species (Jovanović 2007).
In domestic literature, there is almost no data about fungal causes of decline for this ecologically important tree species. Also, research that investigates mycoflora of this tree species is rare even in foreign literature (Farr and Rossman 2016). So far, new research about the occurrence of parasitic and saprophytic species on genus ash (Fraxinus) in this part of the region has mostly been directed to common ash (Fraxinus excelsior L.) (Vemić and Milenković 2018, Karadžić et al. 2019, Vemić 2020) and narrow-leaved ash (Fraxinus angustifolia Vahl) (Keča et al. 2017, Karadžić et al. 2019. Besides multiple benefits of flowering ash (Fraxinus ornus), there is a need for investigation of its mycoses because the other two autochthonous ash species in Europe are endangered by fungus Hymenoscyphus fraxineus (Kowalski) Baral, Queloz & Hosoya (Bakys et al. 2009, Gross et al. 2014, which causes greater ecological pressure to this tree species. The main purpose of the research was to identify the most important parasitic and saprophytic species associated with the decline of flowering ash (Fraxinus ornus) trees in parks of Serbia and Montenegro. This way, knowledge about the decline of flowering ash (Fraxinus ornus) in urban areas is gained and can serve later as a basis for investigation of this tree species' mortality in natural

Field Methods
Field methods included the examination of terrain and taking samples for laboratory analyses. Terrain examination and taking samples was performed in the period of 2017-2022, 2-3 times a year in a range of 3 months. Terrain examination covered all larger cities in Serbia and Montenegro in search for flowering ash (Fraxinus ornus) trees. Special emphasis was put on the cities of Belgrade, Danilovgrad and Cetinje due to a significant number of flowering ash (Fraxinus ornus) trees. All trees with visible changes that can resemble mycosis symptoms or on whom visible fruit bodies of fungi were found were used as a sample for determination of fungi. Samples of symptomatic tissues including leaves, bark and branches were taken from symptomatic trees. Optionally, from these trees, fragments of wood or fruit bodies of macrofungi were collected. For taking samples from trees, a knife sterilized in 96% alcohol was used.

Laboratory Methods
Laboratory methods included determination of fungal species from the collected material. Conventional methods of identification based on morphological characteristics were used for the identification of fungi. Observation of fungal morphology was under enlargement of 400x using Am Scope B120 C E1 microscope. Preparation of temporary histological sections and isolation of mycelium from symptomatic tissues for microscopic analyses were conducted according to Muntanola-Cvetković (1990).

rEsULts
Diversity of recorded fungi on flowering ash (Fraxinus ornus) trees in Serbia and Montenegro is presented in Table 1.
On flowering ash (Fraxinus ornus) trees 21 taxa of parasitic and saprophytic fungi were recorded (Table 1). Whereby, 2 taxa were recorded on leaves, 1 taxon was recorded on root, thin branches and bark, while the remaining 16 taxa of fungi were recorded on the trunk. The most significant fungal taxa found on flowering ash (Fraxinus ornus) in parks of Serbia and Montenegro were Armillaria mellea, Phellinus igniarius aff. and Inonotus hispidus (Table   1, Figure 1, Figure 2). The other taxa that colonized the trunk represented successive species, mostly occurring on trees previously affected with the most significant fungi. The most significant was Schizophyllum commune and somewhat less significant was Meripilus giganteus of these successive species. Succession of fungal occurrence was also expressed on trees damaged by abiotic disorders. Table 2 shows the occurrence of successive fungi according to the condition of substrate.
All taxa of fungi were found in both investigated countries except the species Hymenoscyphus fraxineus, which was found only in Montenegro. Hymenoscyphus fraxineus was recorded only on one tree and it was isolated from necrotic leaf rachis ( Figure 3). Also, necrosis was visible on one young green twig, but it was not isolated ( Figure  3). Taxon Neonectria spp. caused more damage (Figure 3). Based on symptoms and laboratory analyses the fungus was identified up to the genus level.

DIsCUssION
This research showed in detail the diversity of parasitic and saprophytic fungi on flowering ash (Fraxinus ornus) trees in urban conditions of Serbia and Montenegro. The majority of found taxa were recorded for the first time on flowering ash (Fraxinus ornus) in this part of the region. Further molecular analyses are needed to closely identify and confirm all recorded fungal taxa in this study, particularly Ascomycota and Basidiomycota fungi within certain species complexes or those fungi that were impossible to identify to the species level based on morphological characteristics due to old, dry and damaged fruit bodies.
Problems regarding the decline of trees species from genus Fraxinus in Europe culminated through the occurrence of fungus Hymenoscyphus fraxineus. Flowering Pleurotus spp.

Meripilus giganteus
Omphalotus olearius  (Kirisits and Schwanda 2015), although damaging consequences are much less frequent because the fungus is limited to leaves and surrounding tissues (Kirisits 2017). Confirmed presence of fungus Hymenoscyphus fraxineus on flowering ash (Fraxinus ornus) in this part of the region has great importance for understanding the distribution and ecology of this pathogen. Considering that fungus Hymenoscyphus fraxineus causes the decline of leaves on flowering ash (Fraxinus ornus) (Kirisits 2017), being familiar with the diversity of other fungi occurring on them has significant importance in defining decline progress. Spots on the leaves of flowering ash (Fraxinus ornus) in this part of the region were possibly caused by Ascochyta spp. The presence of Venturia fraxini Aderh. which also has two-celled spores, but which are bigger and light brown instead of hyaline spores of Ascochyta spp. (Ellis and Ellis 1985), has not been confirmed for now. Also, fruit bodies of Venturia fraxini are perithecia (pseudothecia), unlike pycnidia of Ascochyta spp., and only the anamorph of this fungus colonizes living leaves (Ellis and Ellis 1985). Fungus Venturia orni Ibrahim, Schlegel & Sieber morphologically similar to Venturia fraxini (Ibrahim et al. 2016) also was not found. This points to further investigation of fungal diversity in natural stands in order to confirm the presence of these species, especially using molecular methods. It is assumed that air pollution in urban areas influenced the absence or markedly reduced presence of these species considering susceptibility of leaf pathogens to the external environment (Kowalski 2013).
Bark necrosis and further damages as a consequence of their development have great significance in urban conditions in reducing ornamental value of trees (Tello et al. 2005). This research showed the presence of Neonectria spp. on the bark of flowering ash (Fraxinus ornus) trees. Difficulties in sampling tissues due to tree cutting and age of necrosis excluded more detailed analyses. Previous research demonstrated that species Neonectria punicea (J.C.) Schmidt Castl. & Rossman caused bark necrosis on common ash (Fraxinus excelsior) in cases where bark had been previously damaged (Karadžić et al. 2020). Since bark damages in urban conditions are frequent due to different causes, it is assumed that this species was also present on flowering ash (Fraxinus ornus) trees in parks of Serbia and Montenegro.
Decay of trees also has great importance in urban conditions (Tello et al. 2005). However, due to their different bioecological characteristics in urban conditions, lignicolous fungi that cause heart rot and have hard fruit bodies are less frequently distributed (Vasaitis 2013). This theory was confirmed by this research, but also, at some level, there were exceptions from this rule. This can be explained due to the proximity of typical forest ecosystems near the investigated park trees.
Finally, pathogenicity tests are recommended for certain Ascomycota fungi recorded in this study, primarily Ascochyta spp. and Neonectria spp. to evaluate their role in flowering ash (Fraxinus ornus) trees' decline in urban conditions. The obtained results enable adequate protection strategies in parks with flowering ash (Fraxinus ornus) trees in this part of the region. Identification of the most important fungal species associated with diseases of trees potentiate taking such protection measures that will decrease or eliminate their presence.

CONCLUsIONs
This study identified 21 taxa of parasitic and saprophytic fungi on flowering ash (Fraxinus ornus) trees in parks of Serbia and Montenegro. Results and conclusions that follow from them can be presented as following: • On flowering ash (Fraxinus ornus) trees 21 taxa of parasitic and saprophytic fungi were recorded. On leaves 2 taxa were found, 1 taxon was found on the root, thin branches and bark, while 16 taxa were found on the trunk. The majority of taxa were found for the first time on flowering ash (Fraxinus ornus) trees in Serbia and Montenegro, especially in urban areas. • Invasive pathogen Hymenoscyphus fraxineus was for the first time recorded on flowering ash (Fraxinus ornus) in Montenegro. This represents one of the southernmost findings of this fungus and has great significance in studying ecology of this fungus. It is considered that Hymenoscyphus fraxineus is also present on flowering ash (Fraxinus ornus) in Serbia due to more favorable ecological conditions for this fungus. • The most significant species were Armillaria mellea, Phellinus igniarius aff. and Inonotus hispidus. The other taxa occurred less frequently or successively, causing smaller damages to trees. • Recommended protection strategies are focused on regulating a mixture of tree species in parks and local forest stands as well as lowering damages of trees because many fungal species found in this research also colonize other tree hosts.

Author Contributions
Author AV designed the research, performed field investigations, laboratory analyses and wrote the manuscript.

Funding
This research has been fully supported by the Ministry of Education, Science and Technological Development, Republic of Serbia under the contract number 451-03-9/2022-14200169.