Taxonomy and pathogenicity of fungi associated with oak decline in northern and central Zagros forests of Iran with emphasis on coelomycetous species

Oak decline is a complex disorder that seriously threatens the survival of Zagros forests. In an extensive study on taxonomy and pathology of fungi associated with oak decline in the central and northern part of Zagros forests, 462 fungal isolates were obtained from oak trees showing canker, gummosis, dieback, defoliation, and partial or total death symptoms. Based on inter-simple sequence repeat (ISSR) fingerprinting patterns, morphological characteristics, and sequences of ribosomal DNA (28S rDNA and ITS) and protein coding loci (acl1, act1, caM, tef-1α, rpb1, rpb2, and tub2), 24 fungal species corresponding to 19 genera were characterized. Forty percent of the isolates were placed in eight coelomycetous species from seven genera, namely, Alloeutypa, Botryosphaeria, Cytospora, Didymella, Gnomoniopsis, Kalmusia, and Neoscytalidium. Of these, four species are new to science, which are introduced here as taxonomic novelties: Alloeutypa iranensis sp. nov., Cytospora hedjaroudei sp. nov., Cytospora zagrosensis sp. nov., and Gnomoniopsis quercicola sp. nov. According to pathogenicity trials on leaves and stems of 2-year-old Persian oak (Quercus brantii) seedlings, Alternaria spp. (A. alternata, A. atra, and A. contlous), Chaetomium globosum, and Parachaetomium perlucidum were recognized as nonpathogenic. All coelomycetous species were determined as pathogenic in both pathogenicity trials on leaves and seedling stems, of which Gnomoniopsis quercicola sp. nov., Botryosphaeria dothidea, and Neoscytalidium dimidiatum were recognized as the most virulent species followed by Biscogniauxia rosacearum.

2 Materials and methods

Sampling, disease symptoms, and isolation of fungi
During a survey from July to November 2017, twigs and branches of oak trees showing external disease symptoms, dieback, wilting, canker, gummosis and twig/branch discoloration, and bark cracking were collected from Zagros forests of five provinces: Ilam, Kermanshah, Kurdistan, Lorestan, and West Azarbaijan.Cross-sections of the samples were examined to classify internal disease symptoms.To isolate fungi, small pieces of disinfested woody tissues (with 70% ethanol for 3 min) were transferred on potato dextrose agar (PDA) supplemented with 100 mg/L streptomycin sulfate and ampicillin and incubated at 20-25°C.Pure cultures were obtained using single spore or hyphal tip methods on tap water agar (2% WA).The isolates were stored on PDA at 4°C.Representative isolates were deposited in the culture collection of the Iranian Research Institute of Plant Protection (IRAN, Tehran, Iran) and the CBS collection of the Westerdijk Institute, Utrecht, The Netherlands.Isolates that were sequenced and studied morphologically are available in Table 1.

Phylogeny
The generated sequences together with the sequences retrieved from GenBank were aligned using Clustal X v. 1.83 (Thompson et al., 1997) or online MAFFT v. 7 and edited manually in BioEdit v.  7.0.0,where necessary.Each locus was aligned separately and the alignments were concatenated with Mesquite 2.75 (Maddison and Maddison, 2023).Both single and combined loci were analyzed by Maximum Parsimony (MP), Maximum Likelihood (ML), and Bayesian Inference (BI).MP was performed using PAUP v. 4.0b10 (Swofford, 2003).ML and BI were carried out through the online CIPRES Science Gateway (Miller et al., 2012) using RAxML-HPC BlackBox v. 8.2.10 (Stamatakis, 2014) and MrBayes v. 3.2.6 (Huelsenbeck and Ronquist, 2001;Ronquist and Huelsenbeck, 2003), respectively.MP analysis was executed according to Abdollahzadeh et al. (2013).Optimal nucleotide substitution models were detected for each locus using MrModelTest v. 2. 3 (Nylander, 2004).The ML and Bayesian analyses were executed as described by Abdollahzadeh et al. (2020).Phylograms were plotted using FigTree v. 1.4.3 and edited in Adobe Illustrator CS2 v. 12.0.0.A l i g n m e n t s a n d tr e e s w e r e d e p o s i t e d in T r e e B A S E (www.treebase.org;S30794, S30795, and S30796) and taxonomic novelties registered in MycoBank (Crous et al., 2004).

Phenotypic and microscopic studies
Depending on the fungal morphological group, culture characteristics and microscopic fungal structures were examined and recorded from cultures grown on PDA, oat meal agar (OA), malt extract agar (MEA), and yeast malt agar (YMA) at room temperature.Sporulation and fruiting body production were induced under a combination of near-UV and cool-white fluorescent lights under 12-h light/12-h dark conditions.The structure and dimensions of microscopic features were determined and measured in 100% lactic acid or distilled water using an Olympus DP72 camera on an Olympus BX51 microscope and a Cell Sense Entry measurement module.To compute the dimensions of each fungal structure, mean, standard deviation, and 95% confidence intervals were estimated based on at least 30 microscopic measurements.Dimensions are presented as the range of measurements with extremes in brackets followed by mean ± standard deviation.

Pathogenicity tests
Pathogenicity tests of recognized species were performed on leaves and stems of 2-year-old oak (Q.brantii) seedlings in Petri plates and under greenhouse conditions, respectively.The surface of leaves and stems was disinfested with 70% ethanol.Small pieces of bark (≈ 3 × 3 mm) were cut from the stems of potted seedlings and small scratches (≈3 mm) were made on the leaves.Mycelial plugs of 7-d-old colonies on PDA were inoculated on wounded leaves and stems.Controls were inoculated with sterile PDA plugs.To evaluate the phytotoxic activity of fungal isolates, inoculated leaves were incubated at 25°C for a period of 7 days.The inoculated seedling wounds were wrapped with Parafilm and placed under greenhouse conditions (22-28°C) and watered as needed.As the seedlings declined, the external symptoms were recorded and the extent of vascular discoloration (lesion length) was measured.To confirm Koch's postulates, fungal isolates were re-isolated from inoculated leaves and stems on PDA at 25°C and morphologically reexamined.The greenhouse trials were performed as a completely randomized nested design (CRND) with three replications per treatment.To determine differences in lesion lengths caused by inoculated fungi, one-way ANOVA was executed.Homogeneity of variance and normality assumption were examined using Bartlett and Shapiro-Wilk's tests, respectively.Least significant difference (LSD) values were calculated (p = 0.05) using SAS v. 9.1.3.

Disease symptoms, fungal isolates, and species identification
A total of 462 fungal isolates were collected from oak trees (Q.brantii, Q. infectoria, and Q. libani), of which 184 isolates (40%) were accommodated in coelomycetous Dothideomycetes.Various external and internal symptoms (Figures 1, 2) were observed on trees' and twigs' cross-sections listed in Table 1.Primarily, borer feeding sites (e.g., Buprestidae) were observed in association with irregular and central wood necrosis and no correlation was found between fungal species and type of disease symptoms.
According to DNA profiles generated with M13 primer (data not shown), 36 isolates as representatives of recognized DNA banding patterns were selected for morphological and phylogenetic analyses.
The first DNA sequence dataset (ITS: 71, tub2: 47 sequences) consisted of our selected isolate IRAN 4323C, 68 isolates belonging to 66 species of the Diatrypaceae family, and two outgroups Xylaria hypoxylon CBS 122620/Kretzschmaria deusta CBS 826.72.The aligned datasets of ITS (691) and tub2 (454) were combined and subjected to MP, ML, and BI.After alignment, the combined dataset consisted of 1,145 characters including alignment gaps.Of these, 490 were constant, 192 were variable and parsimony-uninformative, and 463 were parsimony-informative.MP analysis of the remaining 463 parsimony-informative characters resulted in 729 most parsimonious trees (TL = 2,925, CI = 0.41, RI = 0.6, HI = 0.59).MrModelTest revealed that the general time-reversible model of evolution (Rodrıǵuez et al., 1990), including estimation of invariable sites and assuming a discrete gamma distribution (GTR+I+G) with six rate categories (lsetnst = 6, rates = invgamma) and dirichlet (1,1,1,1) base frequencies, is the best nucleotide substitution model for both loci (ITS, tub2).The Bayesian analyses of the concatenated alignments of two loci generated 3,682 trees from which 920 trees were discarded as burn-in.The consensus tree and posterior probability values (PP) were calculated from the remaining 2,762 trees.The average standard deviation of split frequencies was 0.009962 at the end of the run.The RAxML search of the dataset with 760 distinct alignment patterns produced a best-scoring ML tree (lnL = −14,040.126555).The ML and Bayesian phylogenetic trees were mapped on the MP tree shown in Figure 5 with MP/ML/BI bootstrap support and posterior probability values at the nodes.In these analyses, our isolate IRAN 4323C was placed in a distinct clade in Alloeutypa, a new genus recently introduced in Diatrypaceae, which is recognized here as a new species named Alloeutypa iranensis sp.nov.close to A. flavovirens CBS 272.87 and isolate MFLU 19-0911 (Figure 5). A. iranensis showed remarkable differences in nucleotide sequences with A. flavovirens CBS 272.87 (ITS: 7 substitutions; tub2: 7 substitutions, 21 deletions/ insertions) and MFLU 19-0911 (ITS: 5 substitutions; tub2: 6 substitutions, 25 deletions/insertions).It is also significantly distinct from the type species A. milinensis FSATAS 4309 based on ITS (12 substitutions, 1 deletion/insertion) and tub2 (16 substitutions, 25 deletions/insertions) sequence data.Isolate MFLU 19-0911 is placed in a distinct clade and is apparently a representative of a new Alloeutypa species.
The second dataset consisting of DNA sequences of six loci (LSU, ITS, rpb2, act1, tef-1a, and tub2) was analyzed to examine the taxonomic position of our Cytospora isolates.New generated sequences were aligned with available authentic sequences of Cytospora species and Diaporthe vaccinii CBS 160.32 as an outgroup (LSU: 43, ITS: 47, rpb2: 41, act1: 44, tef-1a: 39, tub2: 29 sequences).The concatenated alignment of LSU (803), ITS (593), rpb2 (664), act1 (311), tef-1a (420), and tub2 (503) was subjected to MP, ML, and BI.After alignment, the dataset consisted of 3,295 characters including alignment gaps.Of these, 2,283 were constant,  The third dataset contained three loci DNA sequences (ITS: 31, tef-1a: 23, and tub2: 26 sequences) of our selected isolate IRAN 4313C, 29 Gnomoniopsis species, and Melanconis stilbostoma CBS 109778 as an outgroup.The aligned single gene sequences were concatenated and subjected to MP, ML, and BI.The combined dataset consisted of 1,918 characters (ITS: 606, tef-1a: 370, and tub2: 942) including alignment gaps.Of these, 1,099 were constant, 278 were variable and parsimony-uninformative, and 541 were parsimony-informative.MP analysis of the remaining 541 parsimony-informative characters resulted in three most parsimonious trees (TL = 2,367, CI = 0.55, RI = 0.6, HI = 0.45).MrModelTest revealed that the general time-reversible model of evolution (Rodrıǵuez et al., 1990), including estimation of invariable sites and assuming a discrete gamma distribution (GTR +I+G) with six rate categories (lsetnst = 6, rates = invgamma) and dirichlet (1,1,1,1) base frequencies, is the best nucleotide substitution model for all loci (ITS, tef-1a, and tub2).The Bayesian analyses of the concatenated alignments of three loci generated 4,892 trees from which 1,222 trees were discarded as burn-in.The consensus tree and posterior probability values (PP) were calculated from the remaining 3,670 trees.The average standard deviation of split frequencies was 0.009837 at the end of the run.The RAxML search of the dataset with 976 distinct alignment patterns produced a best-scoring ML tree (lnL = −13,465.491131).The ML and Bayesian phylogenetic trees were mapped on the MP tree shown in Figure 7.Our isolate IRAN 4313C was clustered in a strongly supported clade close to G. paraclavulata CBS 123202 with significant nucleotide differences in all three loci ITS (5 substitutions, 2 deletions/insertions), tef-1a (35 substitutions, 8 deletions/insertions), and tub2 (58 substitutions, 9 deletions/ insertions).Thus, it is recognized as a new species and named Gnomoniopsis quercicola sp.nov.(Figure 7).

Taxonomy
Based on phylogenetic analyses and morphology, eight coelomycetous fungal species were characterized and associated with declined oak trees in the central and northern part of Zagros   Frontiers in Plant Science frontiersin.orgforests in Iran.Of these, four species were recognized as new fungal species for science, which are described here as follows: Alloeutypa iranensis S. Bashiri & Abdollahz., sp.nov.(Figure 8) MycoBank: MB850401.Diagnosis: A. iranensis is the third species to be described in Alloeutypa.Since the type species A. milinensis was described based on the sexual morph and we have only observed the asexual morph, it is only possible to morphologically distinguish A. iranensis from A. flavovirens based on the larger dimensions of conidiomata (0.9-3.9 mm vs. 0.7-1.3mm), conidiophores (12.6 × 3 mm vs. 4.9 × 2.4 mm), and conidiogenous cells (32.4 × 2.6 mm vs. 11.5×2.4 mm) and shorter conidia (28.5 mm vs. 34 mm).

Etymology
iranensis refers to the country where the fungus was first found.

Culture characteristics
Colonies on PDA cottony, dense, white, margin smooth, reverse yellowish-white, reaching 75 mm diameter after 7 days at room temperature in the dark; on MEA flat, white and becoming grayish white with age, margin smooth, reverse grayish, reaching 60 mm diameter after 7 days at room temperature in the dark; on OA cottony, white and becoming grayish white with age, margin smooth, reverse grayish, reaching 90 mm diameter after 7 days at Phylogenetic tree of Cytospora spp.inferred from Bayesian analysis of combined LSU, ITS, rpb2, ef1-a, act1, and tub2 sequence data.MP/ML/BI bootstrap support values and posterior probabilities are shown at the nodes.The phylogenetic tree was rooted with Diaporthe vaccinii CBS 160.32.The new species are in boldface.T Ex-type.

Etymology
Named after Dr. Ghorbanali Hedjaroude, emeritus professor of Tehran University, who significantly contributed to the knowledge of mycology in Iran.

Etymology
quercicola refers to Quercus, the host genus from which the fungus was first isolated.

Culture characteristics
Colonies on PDA cottony, with aerial mycelium, colony margin forming a concentric ring with sparse aerial mycelium, followed by additional rings, creating a lobed rosette-like appearance, buff (21′′′ d) to olivaceous (19′′k) at the center, olivaceous buff (21′′′d) to greenish olivaceous (23′′′i), reaching 90 mm after 9 days at room temperature in the dark; on MEA cottony, with aerial mycelium, spreading out in concentric rings, creating a lobed rosette-like appearance, buff (21′′′d) to whitish, reaching 90 mm after 14 days at room temperature in the dark; on MYA cottony, with aerial mycelium, spreading out in concentric rings, creating a lobed rosette-like appearance, buff (21′′′d) to whitish, reaching 90 mm after 14 days at room temperature in the dark (Figures 11A-C).
In pathogenicity tests under greenhouse conditions, Biscogniauxia rosacearum, B. persica, Botryosphaeria dothidea, G. quercicola, and N. Frontiers in Plant Science frontiersin.orgdimidiatum were the most virulent species, which caused a quick decline and death of inoculated seedlings after 28-30 days.Thus, to evaluate the pathogenicity of these species in a distinct analysis, we recorded the external and internal symptoms and analyzed them after 30 days (Table 2).Pathogenicity of the other examined species were recorded after 60 days and analyzed separately (Table 3).In the first statistical analyses, significant differences were determined in lesion lengths between species (F = 54.22,p < 0.001) and B. rosacearum was the most virulent species and G. quercicola was the most virulent coelomycetous species.Gnomoniopsis quercicola showed high phytotoxic activity on leaves examined under in vitro conditions (Figure 15D).Severe leaf yellowing and defoliation on inoculated seedlings and extended brown wood necrosis in cross-sections of inoculated stems were observed 28-30 days after inoculation under  Frontiers in Plant Science frontiersin.orggreenhouse conditions (Figure 15).Botryosphaeria dothidea and N. dimidiatum showed high phytotoxic activity on leaves (Supplementary Figures 1-4D) and inoculated seedlings showed severe leaf blight and defoliation together with wedge-shaped and irregular wood necrosis in cross-sections of inoculated stems 28-30 days after inoculation (Supplementary Figures 1-4).The second statistical analysis also indicated significant differences in lesion lengths between species (F = 318.33,p < 0.001) and all coelomycetous species; A. iranensis, C. hedjaroudei, C. zagrosensis, D. glomerata, and K. variispora were pathogenic and caused leaf necrosis, blight, and defoliation on oak seedlings under greenhouse conditions 60 days after inoculation.Moreover, cross-sections of inoculated stems showed an intermediate vascular discoloration and irregular wood necrosis (Table 3, Figures 12-14, Supplementary Figures 2, 3).All inoculated pathogens were re-isolated from inoculated leaves and seedling stems, confirming Koch's postulates (Figures 12-15, Supplementary Figures 1-4).
Biscogniauxia (B.rosacearum and B. persica), Neocosmospora (N.metavorans and Neocosmospora sp.), and Cytospora (C.hedjaroudei and C. zagrosensis) were the most prevalent identified fungi associated with oak decline.Among the coelomycetous fungi, members of two well-known families Botryosphaeriaceae (Botryosphaeria and Neoscytalidium) and Cytosporaceae (Cytospora) in association with decline of woody plants were isolated from declined oak trees in the central and northern part of Zagros forests.
Gnomoniaceae, as the second largest family of Diaporthales, is found on leaves and twigs of hardwood trees, shrubs, and herbaceous plants (Walker et al., 2010).Gnomoniopsis Berl., as a distinct genus based on the type species Gnomoniopsis chamaemori (Fr.)Berl., is isolated only from three plant families Fagaceae, Onagraceae, and Rosaceae (Wang et al., 2022).Thus far, several species, namely, G. clavulata, G. paraclavulata, G. daii, G. fagacearum, and G. silvicola, have been recorded on various Quercus species (Sogonov et al., 2008;Walker et al., 2010;Jiang et al., 2021;Jankowiak et al., 2022).To date, Discula quercina is the only Gnomoniaceae species reported from Q. infectoria, Q. macranthera, and Q. rubra in Iran (Khabiri, 1958;Ghasemi-Esfahlan et al., 2019;Hanifeh et al., 2019).In this study, we characterized a new Gnomoniopsis species named Gnomoniopsis quercicola sp.nov.from all three Quercus species in Zagros forests of West Azarbaijan, Kurdistan, Kermanshah, and Ilam provinces.Gnomoniopsis quercicola was placed in a distinct clade close to G. paraclavulata with significant differences in DNA sequence data and growth rate of colony on culture media.This species was the most pathogenic coelomycetous species and the second highly virulent species after B. rosacearum in both pathogenicity experiments on oak leaves and seedling stems.
The majority of identified fungi (19 out of 24, 79%), including all coelomycetous species, were determined as pathogenic; thus, it is important to consider them in various aspects of pathogenicity, host range, geographic distribution, genetic diversity, and management.Since phytotoxic secondary metabolites are major biochemical weapons, as pathogenicity or virulence factors in fungal pathogens causing canker, dieback, and decline diseases in woody plants (Evidente et al., 2019), we have studied and characterized phytotoxic compounds of some species such as B. rosacearum (Masi et al., 2021), F. rabenhorstii (Bashiri et al., 2020a), and S. banihashemiana, previously identified as S. macrostoma (Di Lecce et al., 2020).At the time being, phytotoxic metabolites of six species, A. iranensis, G. quercicola, N. dimidiatum, Ph. tuscanicum, Cosmospora butyri, and Neocosmospora sp., are being investigated.
In this study, branch canker, dieback, and defoliation were obviously dominant external symptoms of declined oak trees accompanied by borer hole and central and irregular necrosis as common internal symptoms on twigs' cross-sections.We found no correlation between fungal species isolated and type of internal and external symptoms as we can infer from several studies (Mohammadi et al., 2014;Hashemi and Mohammadi, 2016;Esparham et al., 2020;Soltaninejad et al., 2017;Bashiri et al., 2022).Most of the identified species were isolated in association with borer hole (feeding site of insects) on infected trees; thus, it is necessary to consider the role of insects in transmission, penetration, and emergence of opportunistic fungal pathogens, weakness, and decline of oak trees in combination with fungal species.In the last decades, many fungi have been isolated from insects or feeding sites of larvae including Biscogniauxia rosacearum, B. persica, B. dryophila, Neocosmospora ewallaceae, N. ambrosia, N. metavorans, Fusarium spp., Diplodia corticola, Dothiorella iberica, and Cryphonectria naterciae (Pazoutova et al., 2010;O'Donnell et al., 2012;Freeman et al., 2013;Bateman et al., 2016;Herr et al., 2016;Bashiri et al., 2022).
According to the composition and diversity of the identified fungal species, the pathogenicity of the majority of species, and the presence of some well-known and new highly virulent species (e.g., Biscogniauxia rosacearum, Botryosphaeria dothidea, N. dimidiatum, and G. quercicola sp.nov.) together with their association with insect borer hole, we strongly recommend to investigate in more detail all identified fungi specifically the most virulent ones and new fungal species to discover their interaction with growing oak species, as well as their relationships together and with insects and abiotic stress.Concurrently, regarding the threats facing Zagros oak forests including drought, heat and dust stresses due to climate change, wildfire, plant diseases (charcoal canker), and pests (borer beetles and green oak tortrix), it is crucial to prevent inappropriate human exploitation and forest management through training and improving welfare of local communities and following environmental friendly approaches dealing with plant diseases and pests.

FIGURE 1
FIGURE 1 External symptoms on oak trees.(A) Defoliation, (B, C) dieback, and (D-F) cankers and discoloration on twigs and branches.

FIGURE 3
FIGURE 3Distribution and frequency of identified fungi at the generic level.

FIGURE 4
FIGURE 4Distribution and frequency of identified fungal species.

FIGURE 12
FIGURE 12 Pathogenicity tests and disease symptoms caused by A. iranensis on oak seedlings in greenhouse (A-C, G) and leaves under in vitro (D, E) conditions.(A) Inoculated plants after 2 months.(B) Control.(C) Necrotic lesion on stems.(D, E) Necrotic spot on leaves and control.(F) A. iranensis colony re-isolated from inoculated seedlings.(G) Stem cross-sections showing wood necrosis and discoloration.

FIGURE 13
FIGURE 13 Pathogenicity tests and disease symptoms caused by C. hedjaroudei on oak seedlings in greenhouse (A-C, G) and leaves under in vitro (D, E) conditions.(A) Inoculated plants after 2 months.(B) Control.(C) Necrotic lesion on stems.(D, E) Necrotic spot on leaves and control.(F) C. hedjaroudei colony re-isolated from inoculated seedlings.(G) Stem cross-sections showing wood necrosis and discoloration.

FIGURE 14
FIGURE 14 Pathogenicity tests and disease symptoms caused by C. zagrosensis on oak seedlings in greenhouse (A-C, G) and leaves under in vitro (D,E) conditions.(A) Inoculated plants after 2 months.(B) Control.(C) Necrotic lesion on stems.(D, E) Necrotic spot on leaves and control.(F) C. zagrosensis colony re-isolated from inoculated seedlings.(G) Stem cross-sections showing wood necrosis and discoloration.

FIGURE 15
FIGURE 15 Pathogenicity tests and disease symptoms caused by G. quercicola on oak seedlings in greenhouse (A-C, G) and leaves under in vitro (D,E) conditions.(A) Inoculated plants after 1 month.(B) Control.(C) Necrotic lesion on stems.(D, E) Necrotic spot on leaves and control.(F) G. quercicola colony re-isolated from inoculated seedlings.(G) Stem cross-sections showing wood necrosis and discoloration.

TABLE 1
Isolates, frequency, location, host, and collection codes of identified fungal species, disease symptoms observed and sequences generated in this study.
T Ex-type.

Outgroup Cytospora hedjaroudei sp. nov. IRAN 4325C Cytospora zagrosensis sp. nov. IRAN 4324C IRAN 4326C
T Cytospora carbonacea CFCC 89947 T Cytospora hippophaes CFCC 89639 T Cytospora friesii CBS 194.42 T FIGURE 6 One of the three most parsimonious trees of Gnomoniopsis spp.obtained from combined ITS, tef-1a, and tub2 sequence data.MP/ML/BI bootstrap support values and posterior probabilities are shown at the nodes.The phylogenetic tree was rooted with Melanconis stilbostoma CBS 109778.The novel species is in boldface.T Ex-type.

TABLE 2
Number of isolates, observed symptoms, and mean lesion length measured in pathogenicity studies of five highly virulent species recorded and analyzed after 30 days.

TABLE 3
Number of isolates, observed symptoms, and mean lesion length measured in pathogenicity studies of 19 species recorded and analyzed after 60 days.
associated with Persian oak (Q.brantii) in Zagros forests located in Kermanshah, Ilam, and Lorestan provinces, but it is reported for the first time from Kurdistan and West Azarbaijan provinces.In this study, B. dothidea and N. dimidiatum were determined as highly virulent in both pathogenicity experiments on leaves and seedling stems of Q. brantii.Pathogenicity of both species on oak has previously been confirmed