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New pathogenic and endophytic fungal species associated with Persian oak in
Iran

Article  in  European Journal of Plant Pathology · August 2019

DOI: 10.1007/s10658-019-01830-y

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Eur J Plant Pathol
https://doi.org/10.1007/s10658-019-01830-y

New pathogenic and endophytic fungal species associated

with Persian oak in Iran
A. Alidadi & M. Kowsari & M. Javan-Nikkhah &
G. R. Salehi Jouzani & M. Ebrahimi Rastaghi

Accepted: 6 August 2019

# Koninklijke Nederlandse Planteziektenkundige Vereniging 2019

Abstract The objectives of the present study were to obtained from ITS, 28S and 18S regions of ribosomal
identify composition of fungal communities associated DNA. Some fungal species such as Neoscytalidium
with healthy and declined oak trees and seedlings in Ilam dimidiatum and Obolarina persica were obtained only
Province, Iran, and to evaluate their role in occurrence of from branches of Persian oak trees with decline symp-
the oak decline. Fungal isolates were obtained from toms. The Acremonium sp., Coniochaeta sp., Cytospora
branches of healthy and declining Persian oak trees and ribis, Fusarium tricinctum, Microsphaeriopsis olivacea
seedlings in Ilam province during summer and autumn and Neoetophoma samarorum species were found only
2014–2015. Fungal species were identified according to in healthy trees as endophytic species. While,
both morphological and molecular characteristics B. mediterrana, Didymella glomerata, Fusarium solani
and Tricothecium roseum were isolated from both healthy
and declined trees. The F. tricinctum and T. roseum spe-
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10658-019-01830-y) contains cies were found in healthy seedlings. However,
supplementary material, which is available to authorized users. D. glomerata was isolated from both healthy and dried
seedlings. The species B. mediterrana, D. glomerata, N.
A. Alidadi : M. Javan-Nikkhah (*) dimidiatum and O. persica showed pathogenicity on the
Department of Plant Protection, College of Agriculture and
Persian oak seedlings in the greenhouse conditions. Fi-
Natural Resources, University of Tehran, Tehran, Iran
e-mail: Jnikkhah@ut.ac.ir nally, it could be concluded that for the first time two
species, D. glomerata and N. dimidiatum, were recorded
as pathogens associated with Persian oak. In addition,
A. Alidadi
Acremonium sp., Coniochaeta sp., C. ribis, F. solani,
e-mail: amin.alidadi@ut.ac.ir
F. tricinctum, N. samarorum and T. roseum were recorded
M. Kowsari (*) : G. R. S. Jouzani for the first time as endophytic fungi on Persian oak trees.
Microbial Biotechnology Department, Agricultural Biotechnology
Research Institute of Iran (ABRII), Agricultural Research,
Education & Extension Organization (AREEO), Karaj, Iran Keywords Didymella glomerata . Neoscytalidium
e-mail: kowsari@abrii.ac.ir dimidiatum . Oak decline . Endophytic fungi . Pathogenic
fungi . Persian oak . Quercus brantii . Zagros forests
G. R. S. Jouzani
e-mail: gsalehi@abrii.ac.ir
Introduction
M. E. Rastaghi
Expert, Forests, Range and Watershed Management Organization,
Tehran, Iran Oaks (Quercus spp.) are the dominant tree species pres-
e-mail: m_ebrahimi_r@yahoo.com ent in Zagros forest in Iran. Zagros forest is one of the
 Eur J Plant Pathol

most important forest area in Zagros mountains located Linaldeddu et al. 2014). For instance, the fungal species
in western parts of Iran and contains about 5.2 billion Botryosphaeria dothidea, Diplodia corticola, D. seriata
hectares of Iran’s forested area (Jazirehi and Ebrahimi and Neofusicoccum parvum were isolated by
Rostaghi 2013; Sagheb-Talebi et al. 2014). Persian oak Linaldeddu et al. (2014) from trunk and branch cankers
(Quercus brantii) covers more than half of the Zagros of declining holm oak (Q.ilex). Luque et al. (2000)
forest area, and is more widespread and frequent than reported Biscogniauxia mediterranea, Botryosphaeria
other species (Bordbar et al. 2010; Jazirehi and stevensii and Ophiostoma quercus from stems of cork
Ebrahimi Rostaghi 2013; Hassanzad Navroodi et al. oak (Q. suber) with decline symptoms. Furthermore, six
2015). Ilam province by itself comprises about species including B. mediterranea, B. corticola,
641,000 ha of Zagros forest, and the main tree species Cytospora sp., Discula quercina, Fusicoccum sp. and
in these forests is Persian oak (Ahmadi et al. 2014). Pleurophoma cava have been reported as causal agents
During the past three decades, occurrence of oak tree of canker and dieback in branches of cork oak in Tunisia
mortality has been frequently reported in many coun- (Linaldeddu et al. 2010). B. mediterranea and
tries such as Austria, Hungary, Italy, United States, Obolarina persica have been also reported as important
Turkey and other regions of the world (Hämmerli and agents of Persian oak decline in Zagros forests
Stadler 1989; Freer-Smith and Read 1995; Thomas and (Mirabolfathi 2013; Mirabolfathi et al. 2013). In addi-
Büttner 1998; Pérez-Sierra et al. 2013; McConnell and tion, Fungal endophytic communities are highly diverse
Balci 2014; Linaldeddu et al. 2014; Frisullo et al. 2018). group of fungi that inhabit in internal tissues of living
Also, the decline and mortality of oaks is one of the most plants without causing any immediate overt negative
important disease of oak trees in western regions of Iran. effects and present in many kinds of plants (Bacon and
It has resulted in destruction of oak trees in Zagros forest White 2000; Strobel and Daisy 2003; Hyde and Soytong
ecosystems. The disease is widespread in all forests of 2008). Many studies have been performed to determine
Zagros from north to south especially on the southern the diversity of endophytic fungi associated with oak
side of the mountain in Ilam, Lorestan, Kohgilouyeh va trees worldwide (Faeth and Hammon 1997; Gennaro
Boyer-Ahmad, Fars and Kermanshah provinces (Fattahi et al. 2003; Ghobad-Nejhad et al. 2017). In addition,
1994; Ahmadi et al. 2014; Mirabolfathi 2013). Symp- the fungal species Deniquelata quercina, Cytospora sp.,
toms on affected oak trees include significant mortality M. olivacea, Diatrype spp., Leptosphaerulina spp.,
in all parts of the crown, brown-black discoloration of Comoclathris sp. and Thyrostroma sp. have been report-
bark and dead branches. Therefore, production of Per- ed as endophytic fungi from Persian oak in Iran
sian oak seedlings in nurseries and their transplantation (Ghobad-Nejhad et al. 2018; Alidadi et al. 2019). Nev-
to forests is a strategy for restoration of damaged areas ertheless, endophytes usually survive in a latent phase in
in Ilam Province, though mostly transplanted seedlings healthy tissues for the whole lifetime or for an extended
die after a short time after moving to the forest. period of time, but may turn pathogenic during a change
Oak decline is a complex disease that is generally in environmental conditions and can rapidly spread
caused by interactions of several biotic and abiotic fac- from several infection points during their pathogenic
tors, and the symptoms and causal factors may not be phase (Rodriguez et al. 2008). B. mediterranea can
the same in all regions (Akilli et al. 2013; Linaldeddu spend part of its life-cycle as an endophyte in all the
et al. 2011; Moreira and Martins 2005; Ragazzi et al. aerial organs of the oak plants and then become oppor-
1995). Undoubtedly, biotic stresses play an important tunistic pathogen under water stress conditions. In those
role on occurrence of decline symptoms on their host conditions, B. mediterraneais able to rapidly colonize
plants (Akilli et al. 2013). The role of fungi, as an the xylem and bark tissues, induces necrosis and canker
important factor causing decline, has been evaluated in formation (Anselmi et al. 2000; Mazzaglia et al. 2001;
different regions of the world, and a high numbers of Linaldeddu et al. 2011; Safaee et al. 2016).
studies have tried to determine the diversity of fungi The objectives of the present study were to: a) isolate
associated with oak decline symptoms worldwide and characterize the fungal species associated with healthy
(Kowalski 1996; Bruhn et al. 2000; Luque et al. 2000; tree and dead branches of Persian oak trees with decline
Thomas et al. 2002; Balci and Halmschlager 2003; symptoms in the Zagros region, b) Isolate and characterize
Ragazzi et al. 2003; Kelley et al. 2009; Henriques fungi from healthy and dead Persian oak seedlings (in
et al. 2012; Akilli et al. 2013; Mirabolfathi 2013; nurseries and transplanted to the forest); and c) to
Eur J Plant Pathol

determine the pathogenicity of the isolated fungal species Organization of Iran 2013). Several symptomatic sam-
on Persian oak seedling in greenhouse conditions. ples were collected and placed in separate paper bags,
recorded their information and then transferred to the
mycology Lab. The samples were washed under running
Material and methods tap water in order to remove dusts or other surface
contaminations, and subjected to air to be dried at room
Sampling and isolation of fungi temperature for 2–3 h. In order to eliminate epiphytic
contaminations, woody samples were disinfested by the
Sampling was done from healthy and dead branches of ethanol 70% for 30 s and by the 1% sodium hypochlorite
Persian oak trees with decline symptoms in different for 1 min, and rinsed twice by sterile water. To isolate the
forest areas of Ilam province (Forest area including associated fungi, the samples were further cut into small-
Arghavan, Tange Dalab, Chagha Sabz in Ilam County er segments (about 5 mm) in sterile conditions and paced
and Forest area in Malekshahi County) as well as healthy on 2% Water Agar (2% WA) as well as Potato Dextrose
and dead seedlings in nurseries (located in Ilam province, Agar (PDA) and then incubated at 25 °C for 5–7 days.
Eyvan city) and transplanted seedlings in the forests Fungi purification was performed by transferring single
during the summer and autumn of 2014–2015 (Fig. 1). spores and/or single hyphal tips grown on 2% Water
The selected regions are the most affected area by oak Agar (2% WA) onto PDA. The purified isolates were
decline (Forests, Range and Watershed Management stored on PDA slants at 4 C for future studies.

Fig. 1 The map of the Persian

oak sampling locations in the
present study (Ilam province)
 Eur J Plant Pathol

Morphological and molecular identifications to field capacity. The age of the seedlings for the
experiment was 24 months and the mean plant size
Morphological identifications were conducted on gen- was 20–25 cm in height with a 4–6 mm stem diam-
eral culture media, including Potato Dextrose Agar eter at the time of inoculation. The seedlings stems
(PDA), Oat meal Agar (OA), Malt Extract Agar were surface-sterilized in 10% sodium hypochlorite
(MEA), Carnation Leaf Agar (CLA) and Synthetic Nu- for 10 min and artificially wounded (size of wound
trient Agar (SNA). Macro- and micro-morphological 4–5 mm). Inoculations were conducted by placing a
characteristics, such as colonies morphology, color and 1-week-old 5-mm agar plug from each fungal cul-
diameter, and sexual and asexual states characteristics ture and the wounded stems were wrapped with
were recorded and compared with the literature. Total parafilm. Control plants were treated similarly with
genomic DNA was extracted according to the protocol sterile PDA plug. All inoculated and non-inoculated
previously described by Zhong and Steffenson (2001). seedlings were incubated at 25 °C. The inoculated
PCR amplifications were done with the primer pairs plants examined after 1–2 months of incubation and
NS1/NS4 for 18S and ITS1/ITS4 for ITS-rDNA inspected for symptoms development. Each plant
(White et al. 1990), and LROR/LR5 for 28S nrDNA was cut longitudinally through the point of inocula-
(Rehner and Samuels 1995). Generated sequences were tion and the extent of lesion and vascular discolor-
observed and edited in BioEdit V. 7.2.5 (Hall 1999) and ation was measured upward and downward from the
were subjected to BLAST search tool in the GenBank point of inoculation. The statistical analysis of data
nucleotide database. Required sequences for phyloge- was performed using a one-way analysis of variance
netic analysis were retrieved from GenBank and multi- (ANOVA), and the means were compared using
ple sequence alignments were generated using the Duncan’s test by SPSS for Windows version 17
MAFFT version 7 web tool (https://mafft.cbrc. (Chicago, SPSS Inc.) Small pieces of necrotic tissue
jp/alignment/server/) (Katoh and Standley 2013). Phy- from the edge of each lesion were cut and placed on
logenetic estimates were evaluated using the Maximum PDA in an attempt to recover the inoculated fungus
Parsimony Analyses (MP) in PAUP version 4.0b10 and complete Koch’s postulates.
software (Swofford and Sullivan 2003). Ambiguously
aligned regions were excluded and gaps were treated as
missing data. The trees were inferred using the heuristic Results
search option with TBR branch swapping and 1000
random sequence additions. Descriptive tree statistics Sampling and isolation
for parsimony (Tree Length [TL], Consistency Index
[CI], Retention Index [RI] and Homoplasy Index Totally, 120 samples from branches of healthy/declining
[HI]) were calculated for trees generated under dif- Persian oak trees in different forest areas and Persian
ferent optimality criteria. Maximum parsimony oak seedlings in the nursery and forest were collected.
bootstrap values (MP) equal or greater than 50% Finally, 264 fungal isolates were obtained from
are given below or above each node. All identified branches of trees and seedlings. From that, 164 isolates
species have been deposited in the Microbial of were from trees branches (97 isolates from healthy and
Agriculture Biotechnology Research Institute of Iran 67 isolates from declining trees) and 100 isolates asso-
culture collections (ABRII CC). ciated with seedlings (70 isolates from healthy and 30
isolates from died seedlings) (Figs. 2, 3 and 4).
Pathogenicity tests
Morphological identifications
To perform pathogenicity test, two-year-old cork
oak seedlings obtained from the field-collected All of the recovered isolates were investigated on the
acorns (from healthy Persian oak trees in Ilam Prov- basis of morphological features of asexual or very
ince, Eyvan city). The acorns were sown in plastic rarely sexual reproduction stages, and finally were
bags containing peat vermiculite mixture in a 1:1 grouped in 12 morpho-types based on the similarity
ratio. The plants were kept in a greenhouse with of their micro- and macro-morphological charac-
temperature 25–35 °C and irrigated twice a week teristics. Finally, ten fungal species, including
Eur J Plant Pathol

Fig. 2 The flowchart showing the process of sampling from healthy/declined Persian oak trees/seedling

B. mediterrana, C. ribis, D. glomerata, F. solani, isolates was recovered with the highest frequency
F. tricinctum, M. olivacea, N. samarorum, N. from healthy/died Persian oak seedlings (57.14%
dimidiatum, O. persica and T. roseum were identi- and 100%, respectively) (Fig. 5).
fied. In addition, Acremonium sp., and Coniochaeta sp.,
were identified at the genus level. Table 1 reports a Phylogenetic analysis
summarized information about morphological charac-
teristic of the identified species in the study. The total number of 21 newly recovered isolates
The information about relative abundance and origin representing 12 genera, 10 species were selected for
of the isolates was included in the Table 3. The phylogenetic reconstruction using the ITS-rDNA se-
N. dimidiatum and O. persica were obtained only from quences. The newly obtained sequences were deposited
Persian oak trees with decline symptoms, whereas in the GenBank (Table 3). The Maximum parsimony
Acremonium sp., C. ribis, F. tricinctum, M. olivacea trees consisted of two main clades including clad 1
and N. samarorum only from healthy Persian oak trees (Dothideomycetes) and clade 2 (Sordariomycetes) with
as endophytic species. However, the B. mediterrana, D. 53% and 93% bootstrap support, respectively (Fig. 6).
glomerata, F. solani and T. roseum were isolated from The clade 1 included three groups A, B and C, which
both healthy and declining trees. In addition, belonged to the taxonomic families of fungi including
D. glomerata, F. tricinctum and T. roseum were isolated Botryosphaeriaceae, Didymellaceae,
from healthy seedlings, while D. glomerat was isolated Leptosphaeriaceae, respectively. The isolates obtained
from all died seedlings in the nurseries and from the in the present study including N. dimidiatum (isolate:
seedlings transported to the forest area (Table 2). There- 112RA1), D. glomerata (N43–1, N74–1, N42, N59–2,
fore, it could be concluded that D. glomerata was ob- N29–2 and 66SA2), M. olivacea (isolate: 22SA) and
served in all healthy/declining trees and seedlings N. samarorum (isolate: 88SA1) were located in these
(Fig. 5). B. mediterranea showed the highest frequency groups. However, ITS-rDNA sequences were not able
in healthy/declining Persian oak trees (20.61% and to separate species of Neoscytalidium from each other
37.31%, respectively) whereas the D. glomerata (Fig. 6, clade 1-A). Therefore, phylogenetic analysis for
 Eur J Plant Pathol

Fig. 3 The morphological characteristics of six fungal species Coniochaeta sp. isolate 92SA1, h-I: the C. ribis isolate 38SA1,
isolated in the present study, including a-b: the Acremonium sp. j-n: the D. glomerata isolate N42 and o-p: the F. solani isolate
isolate 97SA, c-d: the B. mediterranea isolate 17SA, e-g: the 27RF

Neoscytalidium spp. was performed based on the com- the phylogenetic analysis was performed by using
bined analysis of the ITS, 18S and 28S nrDNA dataset TEF-1α gene for Fusarium spp. isolates. The isolates
by MP method and the results showed that the isolate were identified as F. solani sensu stricto (Fig. 2,
112RA1 clustered with N. dimidiutum strains and is supplementary material). The O. persica (isolate:
distinct from the isolates of N. novaehollandiae (Fig. 66SA) and B. mediterranea (isolates: 17SA,
1, supplementary material). 116SA1 and 60RP1) were located in the clade 2-E
Twelve isolates obtained in the present study were that consisted of members of Xylariaceae family
placed in the clade 2, which included seven groups D (Fig. 6, Clade 2-E). The isolates of the clade 2-F
to J. The clade 2-D comprised of members of and clade 2-G were related to incertae sedis family.
Nectriaceae family. F. solani species complex (iso- The Acremonium sp. (isolate: 97SA) and T. roseum
lates: 27RF and 113SA) and F. tricinctum (isolates: (isolate: 115SA) were placed in these clades, respec-
60B11 and 56R3) were clustered to related taxa in tively (Fig. 6, Clade 2-F and G). The isolates belong-
this clade (Fig. 6, Clad 2-D). To determine the accu- ing to Valsaceae and Coniochaetaceae families were
rate taxonomic position of F. solani species complex, located in the clade 2-H and I, respectively.
Eur J Plant Pathol

Fig. 4 The morphological characteristics of six fungal species N. samarorum isolate 88SA1, i-l: the N. dimidiatum isolate
isolated in the present study, including a-b: the F. tricinctum 112RA1, m-n: the O. persica isolate 66SA and o-p: the
isolate 60B1, c-e: the M. olivacea isolate 22SA, f-h: the T. roseum isolate 115SA

Pathogenicity tests different from the control (Fig. 8a). Also, 70% of inoc-
ulated seedlings were died two months after inoculation.
Pathogenicity tests were conducted using various iso- These pathogenic fungi were re-isolated from the lesion
lates of each species in six replicates. The D. glomerata areas, and the identity as D. glomerata species was
isolates, including N43–1, N74–1, N42, N59–2, N29–2 confirmed by the morphological characterization. The
and N-43 were tested on stems of Persian oak seedlings. pathogenicity tests using selected isolates of
Typical symptoms, including longitudinal lesion on the N. dimidiatum (112RA1, 38SA3, 41SA2 and 59SA3)
inoculation point with dark brown discoloration of resulted in sooty necrotic lesions that developed in both
wood were observed 45 days after inoculation. All the upwards and downwards directions from the inoculation
isolates of Didymella were able to grow in the xylem points within 30 days after inoculation (18–35 mm long)
surrounding the inoculation point on stem of Persian and slight bark peeling was observed in the inoculated
oak (Fig. 7a-f). The length of necrosis in inoculated area and the black fungal spores appeared under the bark
seedlings ranged from 12 to 26 mm in length, and all on the canker surface (Fig. 7g-k). All the strains of
the strains produced lesions significantly (P < 0.05) N. dimidiatum produced significantly (p < 0.05)
Table 1 Morphological characteristics of the identified species in the present study

Fungus name No. of Colony characters Main morphology futures Figure

isolates

Acremonium sp. 3 White/light orange in upper/reverse on the MEA Phialides simple and slender/Conidia one-celled, hyaline and mostly Fig. 3a-b
aggregated in slimy heads at the apex of each phialide
Biscogniauxia mediterranea 45 white at first, becoming gray on upper side and black on The conidiogenous structures were Nodulisporium-like, smooth, Fig. 3c-d
reverse side after seven days on PDA ellipsoid to lemon-shaped
Coniochaeta sp. 5 Olivaceous in center and white at margin on PDA. The conidia were hyaline, allantoid, aseptate that formed into spherical Fig. 3e-g
to ovoid picnidia
Cytospora ribis 15 light orange on the OA Black, globose to ovoid perithecia that covered with dark-brown, Fig. 3h-i
straight setae/Conidia 1-celled, hyaline, smooth-walled, allantoid
and formed on denticles
Didymella glomerata 80 Cottony, brown at center, white at margin on MEA Pycnidia subglobose, solitary or confluent, papillate/Conidia hyaline, Fig. 3j-n
variable in shape, mostly elliptical to ovoid-ellipsoidal, cylindrical,
with polar guttules/Chlamydospores unicellular or multicellular,
solitary or in short chains (2–6 chlamydospores)
Fusarium solani 15 Arial mycelia sparse and white to cream on PDA Macroconidia relatively wide, straight to slightly curved, 3- to 7- Fig. 3o-p
-septate
Fusarium tricinctum 35 Arial mycelia initially white, then become red to pink with Macroconidia slender and falcate to lunate with 3–5 Fig. 4a-b
age on PDA septate/Microconidia oval, fusiform and occasionally napiform with
0–1 septa
Microsphaeriopsis olivacea 12 Olive green on PDA Conidiomata pycnidial/Conidiogenous cells hyaline, subcylindrical to Fig. 4c-e
doliiform/Conidia solitary, initially hyaline and becoming pale
brown, aseptate
Neosetophoma samarorum 4 Pale green at center, slightly darker at margin on PDA Conidiomata pycnidial, unilocular, 1–3 papillate Fig. 4f-h
ostiole/Conidiogenous cells hyaline doliiform to
ampulliform/Conidia hyaline or pale yellow, subcylindrical, ellip-
soid to fusiform, smooth-walled with truncate base
Neoscytalidium dimidiatum 7 Grew fast on PDA/Reaching approximately 66 mm in di- The mycelium anamorph composed of branched mycelium, septate Fig. 4i-l
ameter in two days at 25 °C. and brown hyphae which disarticulated becomes 0 to 1-septate
arthrospores with various morphology/ Coelomycetous asexual
morph composed of pycnidia with ellipsoid to ovoid conidia
Obolarina persica 12 Upper side dark green in center, yellow in the edge and Ascospores were dark brown, ellipsoid, inequilateral with a germ slit Fig. 4m-n
reverse side were completely dark after 7–10 days on PDA around the spore
Trichotecium roseum 31 Orange in center, pale orange to white at margin on PDA Conidiophore slender, simple, very long (2–2.5 μm width, up to Fig. 4o-p
200 μm length)/Conidia obovoid or pyriform, initially aseptate and
become 1 septate
Eur J Plant Pathol
Eur J Plant Pathol

Table 2 The abundance, origin and species types of the isolated fungi from healthy and declined Persian oak trees and seedlings

Fungal species Persian oak trees Persian oak Seedlings

Chagha Sabz Arghavan Tange Dalab Malekshahi Nursery Forest

Healthy Declined Healthy Declined Healthy Declined Healthy Declined Healthy Died Healthy Died

Acremonium sp. 1 – – – 2 – – – – – – –
B. mediterranea 8 5 3 7 6 8 3 5 – – – –
C. ribis 4 – 3 – 1 – 7 – – – – –
Choniochaeta sp. 2 – – – 1 – 2 – – – – –
D. glomerata 2 1 1 2 2 – – 2 30 10 10 20
F. solani 3 2 2 1 2 2 – 3 – – – –
F. tricinctum 5 – 3 – 7 – 2 – 10 – 8 –
M. olivecea – – 3 – 3 – 6 – – – – –
N. dimidiatum – 2 – 2 – 3 – – – – – –
N. samarorum 2 – 2 – – – – – – – – –
O. persica – 4 – 1 – 2 – 5 – – – –
T. roseum 2 4 – – 6 – 1 6 10 – 2 –

different mean lesion lengths from the control (Fig. 8b). destruction of the wood texture was observed at the
Inoculated seedlings were dead 30 days after inocula- inoculation points and finally all the inoculated seed-
tion. The morphological characteristics of the isolates lings died. In addition, the O. persica pathogenicity tests
obtained from the inoculated plants coincided with the showed visible brown to black necrotic lesions devel-
inoculated strains, confirming the Koch’s postulates. oped in both upwards and downwards directions from
The isolates 116SA and 17SA of B. mediterranea the inoculation point (17–30 mm) within 45 days after
were used for the pathogenicity test experiments on inoculation. No symptoms were observed on the control
Persian oak seedlings. After 45 days, a complete seedlings. Both pathogens were separately re-isolated

Fig. 5 The share of different

fungi isolated from different plant
sources
 Eur J Plant Pathol

Table 3 List of fungal species isolated from healthy and declined Persian oak trees and seedlings used for phylogenetic analysis

Fungal species Strains Source Location of isolation Culture collection NCBI accession no.

Acremonium sp. 97SA1 HT Chagha Sabz ABRII 10031 MF772440

Biscogniauxia mediterranea 116SA1 HT Tange Dalab ABRII 10016 KY825111
60RP1 DT Arghavan ABRII 10015 KY825110
Cytospora ribis 38SA1 HT Tange Dalab ABRII 10067 KY825112
Coniochaeta sp. 92SA1 HT Chagha Sabz ABRII 10117 MK069644
92SA2 HT Chagha Sabz ABRII 10118 MK069645
Didymella glomerata 66SA2 DT Malek Shahi ABRII 10058 MF563924
N43–1 HS Nursery Ivan ABRII 10062 MF563928
N74–1 HS Nursery Ivan ABRII 10064 MF563930
N42 DS Nursery Ivan ABRII 10061 MF563927
N59–2 HS Nursery Ivan ABRII 10063 MF563929
N29–2 HS Nursery Ivan ABRII 10060 MF563926
Fusarium solani 113SA1 HT Tange Dalab ABRII 10001 KY825096
27RF DT Tange Dalab ABRII 10000 KY825095
Fusarium tricinctum 60B11 HS Arghavan ABRII 10007 KY825102
56R3 HT Malekshahi ABRII 10006 KY825101
Obolarina persica 66SA DT Malek Shahi ABRII 10019 KY825115
Microsphaeriopsis olivacea 22SA HT Tange Dalab ABRII 10047 KY950242
Neoscytalidium dimidiatum 112RA1 DT Tange Dalab ABRII 10066 MK099884
Neoetophoma samarorum 88SA1 HT Chagha Sabz ABRII 10041 KY950236
Trichotecium roseum 115SA HT Tange Dalab ABRII 10054 KY825116

HT, Healthy tree; DT, Declined tree; HS, Healthy seedling; DS, Dead seedling

from the lesions of all seedlings inoculated with seedlings is of particular importance, and many studies
B. mediterranea and O. persica, confirming the Koch’s have been focused on this subject (Peterson and Smith
postulates. Inoculation of different F. solani and 1975; Sutherland et al. 1989; Lilja et al. 1992; Lilja
T. roseum isolates on Persian oak seedlings did not show 1994; Pathak et al. 2015; Pandey et al. 2018; Alidadi
any symptoms after three months. et al. 2018). The present study focused on isolation and
characterization of fungal flora from healthy/declining
Persian oak trees and healthy/dead Persian oak seed-
Discussion lings. N. dimidiatum with 10.44% frequency was isolat-
ed only from trees with declining symptoms and was
The decline and mortality of oaks is one of the most proved a pathogen on Persian oak trees based on path-
important disease of oak trees worldwide. In Iran, it is ogenicity test results (Fig. 5; Fig. 7g-k). This species has
especially serious in the West regions in the Zagros been previously reported from various sources, includ-
forest (Mirabolfathi 2013; Ahmadi et al. 2014). Produc- ing woody plants (Punithalingam and Waterston 1970;
tion of Persian oak seedlings in nurseries and their Sutton and Dyko 1989), soil, human skin and nails
transplantation into forests is taken in the account as (Moore 1988; Punithalingam and Waterston 1970;
an important strategy to restore the damaged areas in the Crous et al. 2006). N. dimidiatum is known as one of
West regions of Iran. Forest nurseries have an important the most important pathogens of plants and has a wide
role in keeping forest lands productive. Nurseries seed- host range. This species has been reported as causal
lings diseases are important from two points of view agent of different plant diseases, such as leaf blight on
including a) Damage to seedlings in the nurseries and b) Sansevieria trifasciata (Kee et al. 2017), shoot blight
transmission of diseases from nurseries to forest areas. and fruit rot in almond trees (Nouri et al. 2018), wood
Therefore, occurrence of diseases on the nursery canker on grapevine (Rolshausen et al. 2013), canker
Eur J Plant Pathol

Fig. 6 The parsimonious tree for the fungal species isolated in the values for maximum Parsimony values higher than 50 are given
present study based on the ITS rDNA sequences of 31 taxa belong above each branch. The ITS sequence of Paecilomyces divaricatus
to Dothideomycetes and Sordariomycetes. The bootstrap support was used as out group

disease in Ficus nitida and F. benjamina (Al-Bedak et al. it was proved in the present study that the D. glomerata
2018), dieback in Mangifera indica (Ray et al. 2010), species has pathogenicity on Persian oak. So, for the
canker disease in Hylocereus spp. (Xu et al. 2018) in the first time, this species was introduced as new pathogen
world. The present study reported the fungus of oak trees. Moreover, it could be concluded that
N. dimidiatum as a pathogen of Persian oak in Iran for latent infection of oak seedlings by D. glomerata
the first time. may cause the spread of the disease among forest areas
The D. glomerata species is a worldwide distributed as it was observed in both seedlings and trees. In
soil fungus, which has been isolated from various addition, considering to the worldwide distribution
kinds of plants as well as inorganic materials, and and wide host range of this species, careful inspection
frequently found in association with symptoms of of nursery products is essential to prevent introduction
blight, leaf spots and fruit rot (Boerema 2004; Moral of the fungus to the forests.
et al. 2018; Thomidis et al. 2011; Aghapour et al. 2009; In both healthy and declining trees, B. mediterranea
Holz et al. 1989). In the present study, this species was was the most abundant with 20.61% and 37.31%, re-
isolated from branches of the healthy and declining spectively. This species reported as an agent of oak
Persian oak trees with frequency of 5.15% and 7.46%, decline and charcoal disease in many regions of the
respectively, and from healthy and died Persian oak world including Portugal, Spain, Italy, Slovenia
seedlings with frequency of 57.14% and 100%, re- (Henriques et al. 2012; Luque et al. 2000; Linaldeddu
spectively (Fig. 5). In addition, by pathogenicity test, et al. 2014; Jurc and Ogris 2006). In addition,
 Eur J Plant Pathol

Fig. 6 (continued)

association of such fungi with charcoal disease in F. solani was another species which was isolated
Quercus spp. trees have been previously reported in from the healthy and declining trees with 7.21% and
deferent region of Zagros forest including Lorestan, 11.94% frequency, respectively (Fig. 5). A brief over-
Ilam, Fars, Kohgiloye VA Boyer-Ahmad and Golestan view on the literature reveals that this species was pre-
Forests in Iran (Mirabolfathi 2013). In this study, viously reported as the causing agent of canker and wilt
B. mediterranea was isolated as an endophyte fungus symptoms on red and coast live oak, associated with
from healthy oak trees as well as from declined trees in branch and trunk cankers on citrus and stem canker on
different sampling areas. O. persica (17.91%) was the Tectona grandis (Vujanovic et al. 1999; Lynch et al.
second most abundant species observed in the declining 2013; Nemec 1987; Huang et al. 2017). However, this
trees, and isolated as a pathogen species only from species did not cause any disease and symptoms on
branches of the Persian oak trees with decline symptoms Persian oak seedlings during three months. T. roseum
(Fig. 5). This species previously have been reported as was obtained from branches of healthy and declining
an important agent causing dying in Persian oak trees in trees with 9.27% and 14.92%, respectively as well as
Ilam province, Iran (Mirabolfathi et al. 2013). from healthy seedlings (17.14%) (Fig. 5). This species
Eur J Plant Pathol

Fig. 7 The results of

pathogenicity tests on Persian oak
seedlings using different
D. glomerata and N. dimidiatum
isolates. a-f: the symptoms
caused by different D. glomerata,
a D. glomerata isolate N43–1, b
D. glomerata isolate N43, c
D. glomerata isolate N29–2, d
D. glomerata isolate N59–2, e- f.
control, g-k: the symptoms
caused by N. dimidiatum isolates,
g inoculation of N. dimidiatum on
oak seedling (right: inoculated
seedling and left: control), h
N. dimidiatum isolate 38SA3, i
N. dimidiatum isolate 112RA1. j-
k control

has shown high antagonistic activities against Diplodia from both Persian oak trees and seedlings, it seems
corticola causal agent of cankers, vascular necrosis and that it could be transferred by vertical transmission,
dieback on various oak species (Campanile et al. 2007). and its antagonistic activity against Persian oak’s
The T. roseum has been risolated also from Maytenus pathogens could be studied in the future. Therefore,
hookeri as an endophyte and antagonist against other this study may be taken in the account as a potential
pathogenic fungi in vitro (Zhang et al. 2010). In the biocontrol agent of oak pathogens.
present study, the pathogenicity of this species was Finally, it could be concluded that four species, in-
not observed on Persian oak seedlings and did not cluding B. mediterranea, D. glomerata, N. dimidiatum
show any symptoms. As these species was isolated and O. persica, were pathogens on Persian oak. To the

a b
30
35
a
25 a
a 30
Lesion Length (mm)

b
Lesion Length (mm)

20 b 25 b
c c c
20
15
d
15
10
10
e
5 5 d

0 0
N43-1 N43 N59.2 N29-2 N74-1 N42 Control 112RA1 38SA2 59SA3 41SA2 Control
Strain Strains

Fig. 8 Average lesion length (in millimeters) resulting from inoc- error. The letters above bars indicate treatments with significant
ulation of (a) Didymella glomerata, (b) Neoscytalidium difference (P < 0.05). Note: the control showed only faint
dimidiatum strains. The vertical bars represent means standard discoloration
 Eur J Plant Pathol

best of our knowledge, this is the first time that two in Iran. Phytotaxa, 405(4), 187–194. https://doi.org/10.11646
/phytotaxa.405.4.2.
species, N. dimidiatum and D. glomerata, are intro-
Anselmi, N., Mazzaglia, A. & Vannini, A. (2000) The role of
duced as pathogens of Persian oak species. In addition, endophytes in decline of oak species in Italy (A. Ragazzi &
F. solani and T. roseum were isolated from trees with I. Dellavalle, eds): 131-144. Accademia Italiana di Scienze
decline symptoms but were not pathogenic for Persian Forestali, Firenze.
Bacon, C. W., & White, J. F. (2000). Microbial endophytes (pp.
oak, and they are only associated with declined oak
341–388). New York: Dekker.
trees. In the previous studies, the fungal species Balci, Y., & Halmschlager, E. (2003). Incidence of Phytophthora
Alternaria atra, A. infectoria, A. consortialis, species in oak forests in Austria and their possible involve-
A. molorum, Chaetomium globosum, Epicoccum ment in oak decline. Forest Pathology, 33(3), 157–174.
https://doi.org/10.1046/j.1439-0329.2003.00318.x.
nigrum, Immersidiscosia eucalypti, Kalmusia
Boerema, G. H. (Ed.). (2004). Phoma identification manual: dif-
variispora, Petriella sordida, Neocamarosporium ferentiation of specific and infra-specific taxa in culture.
obiones and Sordaria fimicola have been also reported CABI Publishing, Wallingford, U.K, pp 497.
associated with declined Persian oak trees in Zagros Bordbar, K., Sagheb-Talebi, K., Hamzehpour, M., Joukar, L.,
Pakparvar, M., & Abbasi, A. R. (2010). Impact of environ-
forests(Alidadi et al. 2018).
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In addition, the Acremonium sp., Coniochaeta teristics of manna oak (Quercus brantii Lindl.) in Fars prov-
sp., C. ribis, F. tricinctum and N. samarorum species ince. Iranian Journal of Forest and Poplar Research, 18(3),
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Pickens, J. B. (2000). Distribution of Armillaria species in
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Acknowledgments The authors would like to thank of Saadi 30(1), 43–60. https://doi.org/10.1046/j.1439-
Karami and Ebrahim Karimi for their help in sampling and the 0329.2000.00185.x.
Agriculture Biotechnology Research Institute of Iran (ABRII) and Campanile, G., Ruscelli, A., & Luisi, N. (2007). Antagonistic
University of Tehran for their financial supports of this projects. activity of endophytic fungi towards Diplodia corticola
assessed by in vitro and in planta tests. European Journal
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