Basilaphelenchus gorganensis n. sp. (Aphelenchoidea,Tylaphelenchinae) from wood from northern Iran

Basilaphelenchus gorganensis n. sp. is described and illustrated from wood and bark of a dead tree from northern Iran. The new species is characterized by female body length (415–559 µm), three‐lined lateral fields, a sclerotized cephalic vestibule and cephalic framework, thin stylet with three elongate backwardly directed knobs, small spherical to oval metacorpus, with small and posteriorly located valve, simple vulva without any flap apparatus, 59‐ to 79‐µm‐long post‐vulval uterine sac, functional rectum and anus and dorsally convex, ventrally concave, usually ventrally bent conical female tail with a sharp tip. Males are common, apparently functional and characterized by well‐curved spicules, three pairs of small caudal papillae and no bursa at tail tip. Molecular phylogenetic inferences using partial sequences of small and large subunit ribosomal RNA genes (SSU and LSU rDNA) from different isolates of the new species revealed it differs from currently sequenced species and belongs to the Tylaphelenchinae clade.     

Molecular and morphological characterization of Ektaphelenchoides maafiae n. sp. (Nematoda: Ektaphelenchinae) from northern forests of Iran

Ektaphelenchoides maafiae n. sp. was isolated during a survey of nematodes associated with bark samples of an oak tree (Quercus castaneifolia) in Gorgan, Golestan Province, northern Iran. The new species has a body length of 480–546 μm (in females) and 431–480 μm (in males). The cuticle is weakly annulated, with three lateral lines. Lip region offset. The stylet is 13–15 μm long without basal swellings. The excretory pore is located at the level of the metacorpus base to slightly posterior, and hemizonid is at 15–17 μm posterior to the excretory pore. The post‐uterine sac is short, 6–8 μm long. Spicules having rounded condylus, rostrum short, conical with bluntly pointed tip, a cucullus (apophysis) presented on the dorsal distal end. Male tail bearing four (2 + 2) caudal papillae, conical, with sharply pointed terminus. The new species is close to four known species of the genus, including E. hunti, E. ruehmi, E. caspiensis and E. poinari, but differs from them by body size, shape of tail terminus, stylet length, shape and size of spicules, length of post‐vulval uterine sac and number of caudal papillae. Phylogenetic analysis based on small subunit (SSU) and partial large subunit (LSU) sequences of rDNA confirmed its status as a new species.     

New morphometric ranges for Bursaphelenchus willibaldi Schönfeld et al. 2006 (Nematoda: Parasitaphelenchidae) from Iran

Bursaphelenchus willibaldi, associated with trees in two separate locations in forests of northern Iran, are characterized and illustrated based on morphological, morphometric and molecular data. The Iranian population of B. willibaldi has a body length of 392–595 μm, stylet length of 12–14 μm, c‐index of 9.0–14.5 and PUS length of 51–82 μm. Males have spicules 15–18 μm long along the arch line. The Iranian population showed morphometric variations compared with the originally described samples. For example, it has a shorter body in females and males, greater c‐index, slightly greater range of V, slightly smaller range of PUS and tail length. Molecular phylogenetic analysis of the recovered populations revealed both sequenced isolates forming a clade with one European isolate of the species using Bayesian inference (BI) analysis with full Bayesian posterior probability (BPP).     

Dothistroma spp. in Western Ukraine and Georgia

Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.     

Morphological and molecular characterization of Leptomelanconium allescheri associated with necrotic lesions on Pinus mugo needles in the Polish Tatra Mountains

Leptomelanconium allescheri was identified for the first time in Poland in 2016 in the Tatra Mts. on Pinus mugo needles. It produced numerous conidiomata on necrotic lesions on live needles. In our study, we provide detailed characteristics of disease symptoms and describe morphology of the structures of the fungus. The shape and size of macroconidia are proven to be more variable in vitro than in situ. The malt extract agar colonies of the fungus obtained from germinating macroconidia are described for the first time. Our observations prove also that, apart from macroconidia, L. allescheri produces in vitro unicellular, hyaline microconidia, 3.5–10 × 1.8–5.0 μm in size, the previously unknown aspect in the development cycle of this species. The BLAST search with ITS and LSU sequences of L. allescheri resulted in the closest match with Cenangium acuum accessions (94%–95% and 99%, respectively). Two gene based, LSU plus ITS, phylogenetic positioning of L. allescheri proves that it belongs to Pezizomycotina, Helotiales. Further, on family level, placement of L. allescheri, as well as its two closest species C. acuum and Piceomphale bulgarioides, remains undefined. The L. allescheri barcode sequences (accession numbers: ITS— MF573935 , LSU— MF573936 , IGS— MF573937 ) and cultures were submitted to NCBI GenBank and CBS Utrecht, The Netherlands culture collection.     

河北省长针线虫新纪录种松长针线虫Longidorus pinus Xu,Ye,Wang & Zhao的形态与分子鉴定

2018年,在河北省保定市园林植物紫荆(Cercis chinensis)根围分离到一种长针线虫。经形态学观察和28S rDNA D2D3区序列分析,将其鉴定为松长针线虫(Longidorus pinus Xu, Ye, Wang&Zhao)。其雌虫主要形态特征为:雌虫体长3 048～3 464μm,唇区明显缢缩,宽9.5～10.5μm,侧器囊袋状,齿尖针长66.0～69.5μm,导环距体前端27.5～30.0μm,尾长31～33μm,短圆锥形,尾长与肛门处体宽比值=1.5～1.6。松长针线虫河北种群28S rDNA D2D3区与GenBank数据库的山西种群进行序列比对,相似性为99.2%～99.9%。松长针线虫是河北省长针线虫新纪录种。紫荆是松长针线虫的新寄主。     

An integrative study of Sakia sisanganensis n. sp. (Rhabditida: Tylenchidae) from Sisangan forest,Iran, and new morphological observations for the genus

Further data on the morphology (the lip region characters) and phylogeny of the genus Sakia are presented. The new observations were based on scanning electron microscopy (SEM) and light microscopy (LM). A new species, Sakia sisanganensis n. sp., was recovered from rotten wood of a dead beech tree (Fagus orientalis) in northern Iran, herein described and illustrated based on an integrative approach, that is morphological, morphometric and molecular characters. The new species is characterized by a combination of the following features: fine transverse striae and vestigial single band in the lateral field in SEM. Labial area dorso‐ventrally flattened. Oral region with two concentric hexagonal plates, the inner one apparently elevated. Amphidial openings short, slit‐like. Stylet delicate. Median bulb fusiform to spindle‐shaped with weak valvular apparatus. Spermatheca functional. Tail filiform with faintly pointed tip and males common. The new species was morphologically compared with four known species of the genus, viz., S. alii, S. arboris, S. castori and S. indica, all having indistinct lateral fields. Molecular phylogenetic analyses were performed based on the small subunit ribosomal RNA gene (SSU rDNA). In the Bayesian inference (BI), S. sisanganensis n. sp. with two isolates was strongly supported as a sister taxon of a clade harbouring S. arboris + Lelenchus species. However, in the maximum likelihood (ML) analysis, the new species formed a clade with S. arboris, thus supporting the reciprocal monophyly of the genera Sakia and Lelenchus. Accordingly, the test of monophyly was performed (using Bayes factor) and the results did not reject the monophyly of sakia (i.e., S. sisanganensis n. sp. and S. arboris as sister taxa) based on the currently available data.     

First report on Bursaphelenchus sexdentati (Nematoda: Aphelenchoididae) in Israel

This is the first report and characterization of Bursaphelenchus sexdentati and the first official report of the presence of the genus Bursaphelenchus in Israel. This species was isolated from Orthotomicus erosus (Coleoptera: Scolytinae) found on Pinus halepensis in the Judean foothills in Israel. Nematodes collected from insect galleries were reared on fungal cultures and identified based on morphological diagnostic characters for the genus Bursaphelenchus. Sequencing analyses of the Internal Transcribed Spacer (ITS) region, small subunit (SSU) and large subunit (LSU) of ribosomal DNA confirmed the identification of this nematode species collected from wood and directly from the insect body.     

Characterization of Corynelia uberata Fr., a putative fungal pathogen of Podocarpus falcatus in Ethiopian forests

Corynelia spp. are ascomycetes belonging to the order Coryneliales and are thought to be obligate parasites of trees in the Podocarpaceae. The aims of this study were to determine the disease intensity of Corynelia infection on Podocarpus falcatus in Ethiopian forests and verify the identity of Corynelia spp. from Ethiopia and other countries using morphological and molecular methods. Disease surveys were conducted in P. falcatus forest areas at Adaba‐Dodola, Bushoftu, Menagesha, Shashamane and Wondo Genet in Ethiopia between 2009 and 2011, and samples were collected for morphological and molecular studies. Additional dried specimens morphologically collected as C. uberata, C. portoricensis and C. tropica from Podocarpus species in Kenya, South Africa, Puerto Rico and New Zealand were also characterized. Morphologically, the South African specimen (F‐006479) of C. uberata had significantly larger ascospores when compared with the other specimens. There was a high sequence similarity (99–100%) in the internal transcribed spacer and 5.8S (ITS‐5.8S) region among the studied C. uberata sequences. Cloning and amplification of the insert spanning partial small ribosomal unit (SSU) and ITS‐5.8S regions of ribosomal DNA validated the unidentified ITS‐5.8S region as the sequence of C. uberata by inferring the reference sequence of SSU rDNA of C. uberata in GenBank. Both neighbour‐joining and/or maximum parsimony methods placed ITS‐5.8S and SSU rDNA sequences of Corynelia spp. at the basal position of the clade Eurotiomycetidae. C. uberata was found to be a potential pathogen on leaves, fruits and young stems of P. falcatus in Ethiopia.     

Initial characterization of an unidentified Armillaria isolate from Serbia using LSU‐IGS1 and TEF‐1‐α genes

Armillaria species have a global distribution and play variable ecological roles, including causing root disease of diverse forest, ornamental and horticultural trees. Accurate identification of Armillaria species is critical to understand their distribution and ecological roles. This work focused on characterizing an unidentified Armillaria isolate from a Serbian forest using pairing, sequencing of the partial large subunit and intergenic spacer‐1 regions of rDNA (LSU‐IGS1) and the translation elongation factor‐1 alpha gene (tef‐1α) genes, and phylogenetic analyses. Despite previously obtained LSU‐IGS1 RFLP patterns that matched the newly described North American Armillaria altimontana, pairing tests and phylogenetic analyses of LSU‐IGS1 and tef‐1α sequences clearly demonstrate that the unidentified isolate is not A. altimontana. Based on LSU‐IGS1, Armillaria gallica isolates were polyphyletic, and the Serbian isolate clustered with a subset of European A. gallica isolates within a well‐supported clade (99%). Based on tef‐1α, the Serbian isolate appeared as a separate, well‐supported clade (97%) that was basal to other poorly resolved, polyphyletic clades containing European A. gallica isolates. It is speculated that the unidentified Armillaria isolate from Serbia could represent an evolutionary ancestral state because of its separate, basal position compared with other clades comprising polyphyletic European A. gallica isolates. Alternatively, this unidentified Serbian isolate could represent an unusual hybrid because of its high‐level sequence heterogeneity, represented by multiple two‐nucleotide codes, within tef‐1α. Further characterization is needed to confirm the taxonomic status and ecological/evolutionary significance of this unique, unknown Armillaria isolate from Serbia.     

Genetic uniformity characterizes the invasive spread of Neofusicoccum parvum and Diplodia sapinea in the Western Balkans

In the past decade, trees and shrubs in the Western Balkans region have been damaged by canker and die‐back disease caused by Botryosphaeriaceae species. These pathogens include Neofusicoccum parvum and Diplodia sapinea. In this study, we determine genetic diversity and structure between populations of N. parvum and D. sapinea from Serbia and Montenegro (Western Balkans) using DNA sequence data of the internal transcribed spacer rDNA, translation elongation factor 1‐alpha, β‐tubulin‐2 and microsatellite markers. The relationship of both pathogens was compared for populations from the Continental (CR) and Mediterranean (MR) regions and for isolates of D. sapinea from Cedrus spp. and Pinus spp. Neofusicoccum parvum and D. sapinea were shown to have a low gene and genotypic diversity across the regions and hosts. All genotypes of D.  sapinea found on Pinus spp. were also present on Cedrus spp. The CR and MR populations of both species were found to be only slightly separated from one another by a geographical barrier. Low genetic diversity and dominance of N. parvum and D. sapinea on non‐native trees suggests that these species have most likely been introduced into Western Balkans, possibly through the movement of infected plants.     

The hosts and geographic range of Dothistroma needle blight in Slovakia

The occurrence and distribution of Dothistroma needle blight (DNB) were studied in 2014–2017 around Slovakia. A total of 84 localities, both native and planted, were investigated, and the presence of DNB was confirmed in 73 of them. In all positive locations, symptoms typical of DNB were observed and the Dothistroma species was confirmed using species‐specific primers either from fungal cultures or directly from needles. Both Dothistroma species—D. septosporum and D. pini—were identified. Both species occurred together in 29 locations, only D. septosporum in 42 and only D. pini in two locations. The host range of D. septosporum included 10 pine species and two spruce species. The host range of D. pini comprised the same number of pine hosts but only one spruce species. Five pine hosts, P. aristata, P. coulteri, P. densiflora, P. jeffreyi, P. × schwerinii, and one spruce host P. abies are new hosts species of D. pini. P. densiflora and Picea pungens have earlier been reported to be susceptible for DNB. In this study, D. septosporum was found from both tree species.     

The true nature of Ganoderma in Iran: Taxonomy based on ITS and mtSSU rDNA

The genus Ganoderma Karst. has broad‐spectrum usage in biotechnology, medicine and the food industry. The complexity of the morphology within species has led to uncertain identification in the past, but recent advancements in molecular identification methods have provided scientists with the opportunity to better understand the taxonomy of the species. The present study attempts for the first time to elucidate the distinctiveness of the Ganoderma species growing in Iran concerning those elsewhere in the world based on mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and internal transcribed spacer (ITS) sequence data. The results disclosed that the G. lucidum Karst. samples collected in Iran are more similar to the European Ganoderma species than to the Asian (Chinese) one used in this study.     

Molecular phylogeny and pathotyping of Fusarium solani: a causal agent of Dalbergia sissoo decline

Sissoo (Dalbergia sissoo), commonly known as shisham, is amongst the finest woods of South Asia, but ‘wilt’ disease has caused a rapid decline in this species. The cause of the disease remains uncertain. The aim of this study is to identify the causal agent of the disease and characterize isolates made from diseased trees, based on genomic data and variations in virulence. Samples of infected roots, stems and the ooze exuded from infected trees were obtained from plants showing symptoms in different geographical regions of India for the isolation of microorganisms. Isolates were used to inoculate healthy plants. Based on the morphological characteristics, genus‐ and species‐specific PCR, and in silico analysis of 5.8S rDNA‐ITS regions, of the 38 fungal isolates, 24 and 14 were identified as Fusarium solani and Fusarium sp., respectively. In a pathotyping study, eighteen F. solani isolates, isolated from roots and stem parts of symptomatic plants, induced typical wilt symptoms when inoculated through soil and roots on D. sissoo seedlings of 1–15 months in age. The population of F. solani was the highest in infected roots and the lowest in parts of stems, gradually decreasing with height, and was isolated constantly up to approximately 40% height of the seedling. F. solani isolates used in inoculations were successfully re‐isolated from the rhizosphere, infected roots and wilted stems, as confirmed using isolate‐specific DNA fingerprints. Molecular phylogenies based on rDNA‐ITS sequences showed that the 38 isolates fell into 2 groups. Group I comprised of F. solani isolates from D. sissoo and F. solani sequences in the NCBI GenBank database, whereas group II included Fusarium isolates other than F. solani. These results are helpful in developing integrated control measures for this highly variable pathogen and to establish a base for future population studies.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.