Fungi associated with grapevine trunk diseases (GTDs) with emphasize on pestalotioid species in Kurdistan Province, Iran

Grapevine trunk diseases (GTDs) are destructive and important economically with worldwide distribution. In this survey 233 fungal isolates were obtained from grapevine cultivars showing trunk diseases symptoms in Kurdistan Province, Iran. Based on sequences data and morphology 24 species belong to 20 genera were characterized. Botryosphaeriaceae, Alternaria, Sporocadaceae and Phaeoacremonium members were the most prevalent identied fungal groups. At the species level Botryosphaeria dothidea, Alternaria malorum, Phaeoacremonium aleophilum and Acremonium sclerotigenum were the most frequent identied species. All species are new records in Kurdistan Province. Clonostachys rosea and Neoscytalidium novaehollandiae are new records on grapevine in Iran. Acremonium sclerotigenum, Alternaria chlamydosporigena, Ascochyta herbicola and Paecilomyces formosus are new records on grapevine around the world. In phylogenetic analyses based on LSU, ITS, TEF-1α and TUB2 sequence data four pestalotioid species belong to Sporocadaceae were identied. Of these, three species are new for science and introduced here as Seimatosporium marivanicum, Sporocadus kurdistani and Xenoseimatosporium kurdistanicum. Furthermore, three new combinations in Sporocadus are proposed. both forward and reverse primers using Biosystems 3730xl DNA Analyzer Forward and reverse reads were paired and consensus sequences obtained BioEdit new sequences Three Sporocadus species including Sporocadus lichenicola, Sporocadus rhododendri and Sporocadus rosigena have previously been reported from grapevine. Spo. lichenicola shows 4 bp (substitution), 8 bp (substitution), 81 bp (28 deletion/insertion, 53 substitutions) and 68 bp (13 deletion/insertion, 55 substitutions) differences with Spo. kurdistanicus. TEF-1α and TUB2 sequences are not available for Spo. rosigena, but in LSU and ITS the type of Spo. rosigena (MFLU 16-0239) has 6 bp and 5 bp differences with Spo. kurdistanicus, respectively. In terms of morphology, conidial dimension of Spo. kurdistanicus is similar with Spo. lichenicola, but conidia of Spo. kurdistanicus are 3-euseptate while in Spo. lichenicola they are 3–4-euseptate and occasionally 5-euseptate. Spo. kurdistanicus is Spo. rosigena having conidia No sequences are available for Spo. rhododendri, but Spo. Spo. rhododendri producing larger conidia at the species level multigene phylogeny on LSU, ITS, TEF-1α and TUB2 of ex-type or authentic strains of all species these four genera. Phylogenetic analyses isolates CJA35 and CJA82 This 30 plant eight isolates three new Two species, but Sei. luteosporum and Sei. vitifusiform are the two closest species. Morphologically if we use conidial characteristics Sei. marivanicum can be distinguish from all other Seimatosporium species reported on grapevine. Sei. marivanicum is separated from Sei. luteosporum by having larger conidia. Although conidial morphology of Sei. marivanicum is more similar with Sei. vitifusiform, but number of eusepta and longer appendages can be used to differentiate these two species. Bayesian (PP = 1) and RAxML(BS = 99) analyses within Sporocadus as a new species Sporocadus kurdistanicus. This species is the fourth Sporocadus species reported from grapevine along with Spo. lichenicola, Spo. rhododendri and Spo. rosigena. Phylogenetically Spo. kurdistanicus is well separated from Spo. lichnicola and Spo. rosigena. No sequences were available for Spo. rhododendri but it is possible to distinguish these two species by having larger conidia (18–24 × 6.5–9.5 µm vs. 15.5–20 × 6.5–8.5 µm) in Spo. kurdistanicus. Sporocadus.

As can be concluded from literature usually different basidiomycetous taxa, more often Fomitiporia mediterranea, Fomitiporia punctata and Phellinus igniarius, and ascomycetous species belong to the most important and well-known genera Phaeoacremonium and Phaeomoniella, Diatrypaceae and Botryosphaeriaceae members are identi ed in association with these grapevine trunk diseases around the world (Larignon and Dubos 1997;Mugnai et al. 1999;Armengol et al. 2001;Fischer and Kassemeyer 2003;Trouillas et al. 2010;White et al. 2011;Bertsch et al. 2013;Úrbez-Torres et al. 2014;Fontaine et al. 2016;Fischer and Peighami Ashnaei 2019). In addition to these common and important fungi, several other fungal species have also been isolated from grapevine showing trunk diseases symptoms.
Kurdistan Province located in Iran is a part of Zagros Mountains occupied by early humans and ancient history in agriculture. Oak and grapevine are the two common trees that can be nd growing across the Zagros Mountains. It is the rst research on GTDs in this part of the world. In this survey during 2012-2014 some 230 fungal isolates were obtained. This study aimed to characterize these isolates based on morphology and DNA sequence data.

Sampling and fungal isolation
During a survey between 2012 and 2014 on grapevine trunk diseases in Kurdistan Province, twig and trunk samples of grapevines showing trunk diseases symptoms (cv. Askari, Bidaneh Se d, Farkhi, Rasha and Sahabi) were collected from vineyards all over 10 years old in 25 different villages. Grapevine cultivars showed different symptoms consisting decline, reduced growth, interveinal yellow-brown or red-brown necrotic spots on leaves known as tiger-stripes pattern, spotting berries (black measles), sectorial and central brown necrosis of the trunks. Cross sections of samples were made and sliced to 0.5-1 cm pieces of infected wood. After surface sterilization, (3-4 min in 70% ethanol) four pieces were placed on 9 cm PDA plates supplemented with 100 mg chloramphenicol, streptomycin or tetracycline. Plates were incubated at 25 ± 2 ºC in the dark. Colonies grown from wood pieces were transferred to PDA plates and incubated at 25 ± 2 ºC in the dark. After 1-2 wk conidiomata were formed on PDA plates. To puri cation of the isolates using singlespore method conidia were transferred to tap water agar (2% WA). After incubation at 25°C for 12 h single germinated conidia were transferred to PDA plates. Representative isolates were deposited in the culture collection (IRAN) of the Iranian Research Institute of Plant Protection (Tehran, Iran) and the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands).

Morphology
Colonies were grown on PDA, MEA and OA at 25 ± 2°C for 1-2 wk. Structures were mounted in 100 % lactic acid or water and digital images were recorded with an Olympus DP72 camera on a Olympus BX51 microscope. Measurements were made with the Cell Sense Entry measurement module. For each isolate the mean, standard deviation, minimum and maximum values were calculated from measurements of at least 30 fungal structures. Conidial length was measured from the base of the basal cell to the base of the apical appendage, and conidial width was measured at the widest point of the conidium (Bonthond et al. 2018). Dimensions are presented as a range with extremes and mean ± standard deviation in parentheses. Depending fungal taxonomic groups the colony morphology and growth rate were determined on different culture media and temperature in the dark. For pestalotioid fungi colony morphology and growth rate were determined on MEA and PDA at 21°C in the dark. After 2 wk mycelial growth was measured and cultural characteristics were recorded based on the colour charts of Rayner (1970).
Forward and reverse reads were paired and consensus sequences were obtained using the software BioEdit v. 7.0.0 (Hall 2004). All new sequences were submitted to GenBank (Table 1).  Ronquist 2001, Ronquist andHuelsenbeck 2003) and RAxML-HPC BlackBox v. 8.2.10 (Stamatakis 2014), respectively. The optimal nucleotide substitution models were determined for each locus using MrModelTest v. 2.3 (Nylander 2004). Bayesian analyses were implemented under the optimal nucleotide substitution models with four simultaneous Markov Chain Monte Carlo chains, 10 M generations and a sampling frequency of 1 000 generations, ending the run automatically when standard deviation of split frequencies dropped below 0.01. Burn-in was set to remove 25 % of the rst sampled trees, after which the 50 % majority rule consensus trees and posterior probability (PP) values were calculated. The ML analyses were done using a GTR + GAMMA substitution model and four rate classes with 1 000 bootstrap iterations. The obtained phylogenetic trees were plotted using

Fungal isolates and species identi cation
In this survey some 223 fungal isolates were obtained from grapevines showing trunk diseases symptoms (Fig. 1), which 30 isolates were morphologically pestalotioid belong to Sporocadaceae. Based on morphology and DNA sequence data 24 fungal species belong to 20 genera were identi ed ( Fig. 2; Table 2). All fungal species characterized in this survey are new records for the fungal ora of Kurdistan Province. It is the rst time Clonostachys rosea and Neoscytalidium novaehollandiae are reported on grapevine in Iran. Acremonium sclerotigenum, Alternaria chlamydosporigena, Ascochyta herbicola and Paecilomyces formosus are new records on grapevine around the world. Given that three new pestalotioid species were identi ed for science here we focused on phylogeny and description of the pestalotioid fungi isolated in this study. Based on morphology, cultural characteristics, grapevine cultivar and sampling geographical location 10 out of 30 isolates were selected for sequencing and phylogenetic studies (Table 1).

Phylogeny
Two datasets were subjected to phylogenetic analyses. The rst dataset consisted of concatenated LSU, ITS, TEF-1α and TUB2, containing 55 taxa representing 30 genera and one undetermined clade recognized by Liu et al. 2019 and Lepteutypa fuckelii CBS dataset after alignment contained a total of 3 151 characters (LSU: 823, ITS: 578, TEF-1α: 738, TUB2: 1 000), including alignment gaps. MrModelTest revealed that the general time-reversible model of evolution (Rodriguez 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 (LSU, ITS, TEF-1α and TUB2). The Bayesian analyses of the concatenated alignments of four loci generated 3 672 trees from which 918 trees were discarded as burn-in. The consensus tree and posterior probability values (PP) were calculated from the remaining 2 754 trees. The average standard deviation of split frequencies was 0.009942 at the end of the run. The RAxML search of the dataset with 1 594 distinct alignment patterns produced a best-scoring ML tree (lnL = -37448.896642). The same phylogenetic tree obtained from both RAxML and Bayesian analyses. The posterior probability values (PP) equal to or higher than 0.5 were mapped on the ML tree (Fig. 3). Our isolates were grouped in four genera Seimatosporium, Sporocadus, Truncatella and Xenoseimatosporium. Isolate MFLUCC 14-1196 the type of Seimatosporium italicum placed in Sporocadus clade. Seimatosporium rhombisporum MFLUCC 15-0543 (ex-type) and Seimatosporium ceae MFLUCC 15-0519 (ex-type) constituted a well-supported clade distinct from all other clades. This clade is probably representative of a new genus but as there is only LSU and ITS sequence data available for these two species it is better to generate RPB2, TEF-1α and TUB2 sequences for further phylogenetic analyses to make the nal decision at the generic level.
The second dataset consisted of concatenated LSU, ITS, TEF-1α and TUB2, containing 46 taxa belonging to four genera Seimatosporium, Sporocadus, Truncatella and Xenoseimatosporium and Distononappendiculata banksiae CBS 143906 as outgroup was prepared and analyzed to identify our isolates at the species level. The aligned dataset contained 2 686 characters (LSU: 818, ITS: 515, TEF-1α: 559, TUB2: 782), including alignment gaps. As in the rst dataset, MrModelTest indicated a GTR + I + G as the best t model for all loci (LSU, TEF-1α and TUB2). The Bayesian analyses generated 1 162 trees from which 290 trees were discarded as burn-in. The consensus tree and posterior probability values (PP) were calculated from the remaining 872 trees. The average standard deviation of split frequencies was 0.009786 at the end of the run. The RAxML search of the dataset estimated 1 108 distinct alignment patterns and made a tree with lnL = -17900.721816. Both analyses resulted to the same phylogenetic tree and posterior probability values equal (PP) to or higher than 0.5 were mapped on the ML tree (Fig. 4). In these study based of multigene phylogenetic analyses our isolates placed in four different genera in well supported clades which three of them are recognized as new species for science that are introduced here.
The fourth species, Truncatella angustata is a well-known and common species associated with woody plants. This species has reported from several countries in association with more than 30 woody plant species including Vitis vinifera (Farr and Rossman 2020). Based on the results, Seimatosporium cornii, Seimatosporium italicum and Seimatosporium pseudocornii are transferred to Sporocadus and three new combinations are proposed.

Taxonomy
Based the multigene phylogenetic analyses four pestalotioid species belong to Sporocadaceae associated with grapevine trees showing grapevine trunk diseases symptoms were recognized including Truncatella angustata and three new species along with three new combinations introduced here as follows.

MycoBank MB 838232
Etymology: Name refers to Marivan in Kurdistan Province, Iran where this species was rst found.
Culture characteristics: Colonies on MEA at with uffy aerial mycelium and entire edge, white to buff (19''f), honey (19-21"b) to vinaceous buff (15-17"'d) at the center, reaching 61-64 mm diam after 14 d at 21 °C; on PDA at with uffy aerial mycelium and entire edge, white at the edge to olivaceous grey (21""'i) at the center, reaching 58 mm diam after 14 d at 21 °C.

MycoBank MB838233
Etymology: Name refers to Kurdistan Province in Iran where this species was rst found.

MycoBank MB838234
Etymology: Name refers to Kurdistan Province in Iran where this species was rst found.
All 24 characterized species are new fungal records in Kurdistan Province, Iran. Clonostachys rosea and Neoscytalidium novaehollandiae are reported as new records in association with grapevine in Iran.
Most of the identi ed species have previously been reported on grapevine, but Acremonium sclerotigenum, Alternaria chlamydosporigena, Ascochyta herbicola and Paecilomyces formosus are reported as new records on grapevine in the world.
In a multigene phylogeny based on LSU, ITS, TEF-1α and TUB2 sequences of representative species of all Sporocadaceae genera our representative pestalotioid isolates resided in four different genera Seimatosporium, Sporocadus, Truncatella and Xenoseimatosporium. To recognize our isolates at the species level we performed another multigene phylogeny based on LSU, ITS, TEF-1α and TUB2 sequences data of ex-type or authentic strains of all species belong to these four genera. Phylogenetic analyses showed that two isolates CJA35 and CJA82 are belong to Truncatella angustata. This species has previously been reported from 30 plant species in different countries around the world including Oleae europaea, Quercus brantii, Vitis sp., Vitis vinifera from Iran (Farr and Rossman, 2020). The remaining eight isolates placed in three genera Seimatosporium, Sporocadus and Xenoseimatosporium and identi ed as new species namely, Seimatosporium marivanicum, Sporocadus kurdistanicus and Xenoseimatosporium kurdistanicum. Two isolates IRAN 2300C and IRAN 2310C constituted a distinct and well supported clade (BI-PP/ML-BS = 1/100) in Seimatosporium named as Sei. marivanicum. So far, 10 Seimatosporium species have reported on grapevine namely, Seimatosporium botan, Seimatosporium hysterioides, Seimatosporium lonicerae, Seimatosporium lichenicola (= Sprocadus lichenicola). Seimatosporium luteosporum, Seimatosporium macrospermum, Seimatosporium parasiticum, Seimatosporium vitifusiforme, Seimatosporium vitis and Seimatosporium vitis-viniferae. Phylogentically Sei. marivanicum is clearly distinct from all Seimatosporium species, but Sei. luteosporum and Sei. vitifusiform are the two closest species. Morphologically if we use conidial characteristics Sei. marivanicum can be distinguish from all other Seimatosporium species reported on grapevine. Sei. marivanicum is separated from Sei. luteosporum by having larger conidia. Although conidial morphology of Sei. marivanicum is more similar with Sei. vitifusiform, but number of eusepta and longer appendages can be used to differentiate these two species.
Phylogenetic analyses in this study revealed that Sei. pseudocornii, Sei. italicum and Sei. cornii are belong to Sporocadus, we therefore transferred them to Sporocadu as new combinations. Although asexual morph of Sei. italicum has not seen, as in most of Sporocadus species both apical and basal appendages absent in conidia of S. cornii and S. pseudocornii indicates their taxonomic position in Sporocadus.
Liu et al. (2019)  The type specimens of Sei. pseudoraosae (MFLUCC 14-0468), Sei. pseudorusarum (MFLUCC 14-0466) and Sei. rosigenum (MFLUCC 15-0563) were placed along with strain CBS 113832 in a clade named as Sporocadus rosarum by Liu et al. (2019). Since TEF-1α and TUB2 sequences are not available for Sei. pseudorusarum and Sei. rosigenum and only LSU is available for Sei. pseudorosae the identity of these species is not clear and we thus considered them as intraspeci c variation in Spo. rosarum until these sequence data is available in the future studies.
Xenoseimatosporium kurdistanicum another new species we introduced here is the second species of Xenoseimatosporium a new pestalotioid genus recently introduced by Liu et al. (2019). These two species are easily distinguishable morphologically by conidial dimensions and appendages length as mentioned in the notes under Xen. kurdistanicum.
In a preliminary eld experiment on pathogenicity of some identi ed species on two grapevine cultivars (Bidaneh Se d and Rasha), N. novaehollandiae, B. dothidea and Ph. aleophilum were the most virulent pathogenic species. Four pestalotioid species characterized in this study using an isolate from each species were nonpathogenic, but it is necessary to examine their pathogenicity with more isolates in greenhouse and eld conditions individually and in combination with other species isolated from grapevine in this study.