Morphological and molecular characterisation of <Emphasis Type="Italic">Ditylenchus stenurus</Emphasis> n. sp. (Nematoda: Anguinidae) from western Iran

A new species in the genus Ditylenchus, D. stenurus n. sp. collected from western Iran, is described and illustrated herein based on morphological and molecular studies. The new species is characterised by a body length of 772 (663–863) μm, delicate stylet 6 (5–7) μm long, six lines in the lateral field. Median bulb of pharynx well-developed, muscular with crescentic valve. Post-vulval uterine sac well-developed, 35 (30–45) μm long, female tail elongate-conoid, becoming narrow suddenly with finely rounded terminus. The new species comes close in morphology and morphometrics to five known species of the genus, namely D. arachis, D. caudatus, D. clarus, D. myceliophagus, and D. nanus. DNA sequencing data was obtained on the partial 18S, D2/D3 expansion segments of the 28S rRNA gene and internal transcribed spacer (ITS). The phylogenetic relationships of this species with other Ditylenchus spp. using partial 18S–rDNA and D2/D3 indicated that D. stenurus n. sp. clustered together with several species belongs to the D. triformis-group i. e. D. africanus, D. destructor and D. halictus: all sharing a rounded tail terminus and six lines in lateral fields.     

Characterization of Longidorus helveticus (Nematoda: Longidoridae) from the Czech Republic

Longidorus helveticus was found at two out of 285 sampling sites for the first time in the Czech Republic. Females, males and juvenile stages were analyzed morphologically and morphometrically. The morphological identification of samples was verified by polymerase chain reaction using a species specific primer. Four markers of ribosomal DNA (18S, ITS1, ITS2, D2-D3 expansion segments of 28S rRNA) and two markers of mitochondrial DNA (cox1 and nad4) were sequenced and analyzed and compared with published gene sequences of other populations of L. helveticus. The partial mitochondrial cytochrome c-oxidase subunit 1 gene and partial nicotinamide dehydrogenase subunits 4 gene showed relatively high genetic variation within the species compared with ribosomal DNA markers.     

Molecular and morphological characterisation of <Emphasis Type="Italic">Aphelenchoides paraxui</Emphasis> n. sp. (Nematoda: Aphelenchoididae) isolated from <Emphasis Type="Italic">Quercus brantii</Emphasis> in western Iran

Aphelenchoides paraxui n. sp. is described and illustrated from bark samples of an oak tree (Quercus brantii L.) in Kermanshah province, western Iran. The new species is characterized by body length of 500–660 μm (females) and 630–665 μm (males), lip region set off from body contour, lateral fields with four lines, and total stylet 8–9 μm long with small basal swellings. The excretory pore is located ca one body diam. Posterior to metacorpus valve. The spicules are relatively large (29–33 μm in dorsal limb) with apex and rostrum rounded and well developed and the end of the dorsal limb clearly curved ventrad like a hook. The female tail is conical, the terminus having a complicated step-like projection, usually with many tiny nodular protuberances. Male tail bearing six (2 + 2 + 2) caudal papillae and a well-developed mucro. The new species belongs to the Group 2 category of Aphelenchoides species sensu Shahina (1996) in which eight known species among Group 2–4 sensu Shahina namely: A. arcticus, A. asteromucronatus, A. blastophthorus, A. lichenicola, A. saprophilus, A. seiachicus, A. silvester and A. xui, are the most closed species. Molecular analyses of the partial small subunit rDNA gene (SSU), D2/D3 expansion segments of the large subunit rDNA gene (LSU) and internal transcribed spacer (ITS) revealed this as a new species and supported the morphological results.     

利用28S rDNA D1/D2区和ITS rDNA序列鉴定甜瓜白粉病病原菌

为了明确宁夏干旱带压砂甜瓜白粉病病原菌,从病原菌分生孢子中提取DNA,PCR扩增ITSrDNA和28S rDNA D1/D2区段,测序后进行BLAST比对.结果表明,病原菌的ITS rDNA和28SrDNA D1/D2序列与菜豆叉丝单囊壳白粉菌Podosphaera phaseoli、凤仙花又丝单囊壳白粉菌P.bal-saminae、菊科叉丝单囊壳白粉菌P.fwca、苍耳单囊壳白粉菌P.xanthii、瓜类单囊壳白粉菌P.fuligi-nea等叉丝单囊壳属Podosphaera的多个种的ITS rDNA和28S rDNA D1/D2序列之间相似度均大于99%,鉴定甜瓜白粉病病原菌为叉丝单囊壳属Podosphaera.     

Molecular characterization and diagnostics of stubby root and virus vector nematodes of the family Trichodoridae (Nematoda: Triplonchida) using ribosomal RNA genes

Plant parasitic nematodes of the family Trichodoridae cause substantial yield losses in many agricultural crops. Rapid and accurate identification of trichodorids to the species level is critical for selection of appropriate measures for control. This study analysed 99 sequences of the D2–D3 expansion segments of the 28S rRNA gene and 131 sequences of the 18S rRNA gene from the stubby nematodes belonging to the genera Nanidorus, Paratrichodorus and Trichodorus. Species delimiting was based on the integration of morphological identification, which is not provided in the present article, and molecular‐based phylogenetic inference and sequence analysis. Twenty‐two valid species and several species complexes were identified among nematodes included in the analysis. PCR‐RFLPs of the partial 18S rDNA and the D2–D3 expansion segments of the 28S rDNA were tested and proposed for identification of these nematodes. Gel PCR‐RFLP profiles and tables with restriction fragment lengths for several diagnostic enzymes are provided for identification. Some problems of taxonomy and phylogeny of nematodes of the family Trichodoridae are also discussed.     

Distribution and genetic diversity of root‐knot nematodes (Meloidogyne spp.) in potatoes from South Africa

A molecular‐based assay was employed to analyse and accurately identify various root‐knot nematodes (Meloidogyne spp.) parasitizing potatoes (Solanum tuberosum) in South Africa. Using the intergenic region (IGS) and the 28S D2–D3 expansion segments within the ribosomal DNA (rDNA), together with the region between the cytochrome oxidase subunit II (COII) and the 16S rRNA gene of the mtDNA, 78 composite potato tubers collected from seven major potato growing provinces were analysed and all Meloidogyne species present were identified. During this study, M. incognita, M. arenaria, M. javanica, M. hapla, M. chitwoodi and M. enterolobii were identified. The three tropical species M. javanica, M. incognita and M. arenaria were identified as the most prevalent species, occurring in almost every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu‐Natal, respectively, while M. chitwoodi was isolated from two growers located within the Free State. Results presented here form part of the first comprehensive surveillance study of root‐knot nematodes to be carried out on potatoes in South Africa using a molecular‐based approach. The three genes were able to distinguish various Meloidogyne populations from one another, providing a reliable and robust method for future use in diagnostics within the potato industry for these phytoparasites.     

Control of Fusarium wilt in melon by the fungal endophyte,Cadophora sp.

Two soil-borne fungal endophytes almost completely suppressed the effects of a post-inoculated and virulent strain of Fusarium oxysporum f. sp. melonis when inoculated to axenically reared melon seedlings in Petri dishes. They were identified as Cadophora sp. on the basis of ITS 1–5.8S rDNA–ITS 2 sequences and morphological characters and obtained from the roots of Chinese cabbage grown as bait plants in a mixed soil made up of samples from different forest soils from Alberta and British Columbia, Canada. Hyphae of Cadophora sp. grew along the surface of the root and colonized root cells of the cortex and reduced the ingress of the Fusarium pathogen into adjacent cells. Melon seedlings pre-inoculated with Cadophora sp. were also grown in soil amended with the different N sources, nitrate or the amino acids leucine and valine, and glucose (final C:N ratio?=?10:1). After 4 weeks, these seedlings were transplanted into the field and disease symptoms were assessed. Only the endophyte-inoculated seedlings treated with valine could effectively inhibit the development of Fusarium wilt in two plots and reduced disease symptom development by 43 and 62 %.     

A new stem nematode associated with peanut pod rot in China: morphological and molecular characterization of Ditylenchus arachis n. sp. (Nematoda: Anguinidae)

Surveys conducted in peanut production areas of China revealed peanut pod rot in several fields in Shandong and Hebei Provinces, China. A large quantity of an unknown stem nematode was isolated from the hulls and seeds of peanuts, herein described as Ditylenchus arachis n. sp. The new species is characterized by a combination of the following features: lateral lip sectors distinctly projected, stylet delicate, 8·4–10 μm in length, six lines in the lateral field, tail elongate–conoid, bursa covering about 68–86% of tail length. Pathogenicity tests showed that D. arachis n. sp. could infect peanut (Arachis hypogaea), but not sweet potato (Ipomoea batatas) or potato (Solanum tuberosum). Morphologically, D. arachis n. sp. appears closest to D. africanus, D. myceliophagus and D. destructor, but can be differentiated based upon a combination of morphological characteristics, host preference and molecular sequence data. The results of the phylogenetic analysis, based on 18S rDNA, the D2–D3 expansion region of 28S rDNA, and the ITS1–5·8S–ITS2 region, confirmed its status as a new species. A sister relationship with D. destructor was appointed, rather than with its ecologically very similar congener D. africanus.     

Antifungal activity of <Emphasis Type="Italic">Boerhavia diffusa</Emphasis> L. extract against <Emphasis Type="Italic">Phytophthora</Emphasis> spp. in tomato and pepper

A new longidorid nematode, Longidorus persicus n. sp., is described and illustrated from a population extracted from soil associated with wild rose (Rosa sp.) naturally growing in the mountains close to the village of Cheshmeh-e-Nezamei near the city of Gilan-e-Gharb, Kermanshah province, western Iran. The new needle nematode is characterised by a large body size (6550–7763 μm), an anteriorly flattened lip region, ca 13 μm wide, distinctly set off from body contour by a depression, amphidial fovea large, pocket shaped, slightly asymmetrically bilobed, a moderately long and flexible odontostyle ca 86 μm long, stylet guiding ring located at ca 25 μm from anterior end, posterior arrangement of the pharyngeal gland nuclei, vulva almost equatorial (49–54 %), tail short, about 3/4 of its width, dorsally convex-conoid, with rounded terminus, with a c’ ratio ca 1.3, bearing three pairs of caudal pores and male rare (ratio 1:10 females) with spicules ca 46 μm long. Integrative diagnosis was completed with molecular data obtained using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, and partial 18S-rDNA. The phylogenetic relationships of this species with other Longidorus spp. using D2-D3 expansion segments and partial 18S-rDNA indicated that L. persicus n. sp. clustered together with L. perangustus and L. euonymus: both of them sharing an anteriorly flattened lip region and distinctly set off from body contour by a depression.     

Comparative molecular analysis of Bursaphelenchus vallesianus,a wood‐inhabiting nematode isolated from declining pine trees in the Czech Republic

The Bursaphelenchus genus (Nematoda: Parasitaphelenchidae) comprises mostly wood‐inhabiting nematodes that feed on various tree‐colonizing fungi. One species of the genus, B. xylophilus, has been proven as an agent causing pine wilt disease (PWD). However, involvement of other Bursaphelenchus species in the PWD remains enigmatic. In the current paper, comparative molecular analysis is performed based on nuclear ribosomal DNA (rDNA) of B. vallesianus, a species that was recently isolated from pine trees (Pinus sylvestris) exhibiting wilting and declining symptoms in the Czech Republic. Sequencing of the nuclear‐encoded ITS1–5·8S–ITS2 rDNA region confirmed previous taxonomic conclusions based on morphology. Evolutionary reconstructions resulted in a phylogenetic tree, where the Czech isolate of B. vallesianus occupied a common clade together with other species belonging to the so‐called B. sexdentati group. Unexpectedly, comprehensive analysis of the sequence data revealed a genetic variation distinguishing the Czech isolate of B. vallesianus from all other species of the B. sexdentati group. This dissimilarity consists of the presence of a four nucleotide exchange found in the 5·8S rRNA‐coding gene. The newly identified genetic variation appears to affect the 5·8S rRNA folding, as deduced from secondary structure models. Additionally, it is shown that for the first time, to the authors’ knowledge, both bursaphelenchid internal transcribed spacers (ITS1 and ITS2) fold into the multibranched closed loops. While the ITS2 closed loop is formed with help of canonical 5·8S‐28S rRNA pairing, the ITS1 forms the thermodynamically stable closed loop with no support of flanking rRNA sequences. The current information on bursaphelenchid ITS rDNA sequence diversity and structure is further discussed.     

Morphological and molecular characterisation of Pratylenchus oleae n. sp. (Nematoda: Pratylenchidae) parasitizing wild and cultivated olives in Spain and Tunisia

A new mono-sexual root-lesion nematode species, Pratylenchus oleae n. sp., parasitizing roots of olive plants cv. Koroneiki in commercial fields at Ouled Chamekh (central Tunisia), and wild and cultivated olive (cv. Picual) plants in Agua Amarga (southern Spain) is described. The new species is characterised by the female having a lip region slightly offset and bearing three annuli, stylet 16.5 (14.5-17.0) μm long, with prominent rounded knobs, pharyngeal overlapping rather long (22–36) μm, lateral fields areolated and with four incisures and diagonal lines in middle band, spermatheca rounded but non-functional, tail short, conoid-rounded to subcylindrical, usually annulated terminus, males unknown, and a specific D2-D3, ITS1, 18S-rRNA, hsp90 and COI sequences. Morphologically this species is related to P. cruciferus, P. delattrei, and P. kumamotoensis. The results of the phylogenetic analysis based on sequences of the D2-D3 expansion regions of 28S, partial 18S and ITS rRNA genes confirmed the close relationship of P. oleae n. sp. with P. dunensis, P. penetrans, P. pinguicaudatus, from which was clearly separated. A PCR-based diagnostic assay was also developed for identification of P. oleae n. sp. using the species-specific primers Poleae_fw1_4 and Poleae_rv1 that amplify a 547-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA, which clearly separate from other root-lesion nematodes damaging olive such as P. penetrans and P. vulnus.     

Quantitative detection of <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis> in infected melon roots using real-time PCR

A method was developed for the specific detection, identification and quantification of Monosporascus cannonballus in infected melon roots based on real-time PCR (SYBR® Green chemistry) targeting the ITS1 region of the rDNA conserved between different strains of the pathogen. The specificity of the reaction was assessed using a number of fungi taxonomically and ecologically related to M. cannonballus. The method was highly sensitive and M. cannonballus was first detected in the roots of a susceptible Piel de Sapo cultivar 2 days after inoculation, before symptom appearance. Although conventional PCR methods could also provide such a specific and sensitive detection, real-time PCR was also able to produce reliable quantitative data over a range of 4 orders of magnitude (from 5 ng to 0.3 pg). The method allowed the quantitative monitoring of fungal growth from the very first stages of infection, and was successfully employed in the early screening of resistance. The assessment of disease progress and severity obtained with real-time PCR was more accurate than that obtained with the visual scoring of root lesions or root biomass losses. Therefore, there exists a great potential for its implementation in those steps of breeding programmes where high accuracy is required.     

Identification and characterization of <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler causing <Emphasis Type="Italic">Ceratonia</Emphasis> Blight disease of carob (<Emphasis Type="Italic">Ceratonia siliqua</Emphasis> L.) in Turkey

A new dagger nematode, Xiphinema tica n. sp., is described and illustrated from several populations extracted from soil associated with several crops and wild plants in Costa Rica. The new dagger nematode is characterised by a moderate body size (3276–4240 μm), a rounded lip region, ca 13.5 μm wide, separated from body contour by a shallow depression, amphidial fovea large, stirrup-shaped, a moderately long odontostyle ca 135 μm long, stylet guiding ring located at ca 122 μm from anterior end, vulva almost equatorial (50–54%), well-developed Z-organ, with heavy muscularised wall containing in the most of specimens observed two moderately refractive inclusions variable in shape (from round to star-shaped), with uterine spines and crystalloid bodies; female tail short, dorsally convex-conoid, with rounded end and a small peg, with a c’ ratio ca 0.8, bearing two or three pairs of caudal pores and male absent. The unique and novel uterine differentiation based on the coexistence of a well-developed Z-organ mixed with uterine spines and crystalloid bodies in Xiphinema prompted us to update and include this combination of characters in the polytomous key of Loof and Luc (1990). Integrative diagnosis was completed with molecular data obtained, using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, partial 18S–rDNA and the partial mitochondrial gene cytochrome c oxidase subunit 1 (coxI). The phylogenetic relationships of this species with other Xiphinema spp. indicated that X. tica n. sp. was monophyletic to the other species from the morphospecies Group 4, Xiphinema oleae.     

Detection of Meloidogyne incognita and Pochonia chlamydosporia by fluorogenic molecular probes*

Fluorescent molecular probes were applied for detection of the plant parasitic nematode Meloidogyne incognita and the nematode‐egg parasitic fungus Pochonia chlamydosporia var. chlamydosporia. A region in the M. incognita rDNA including ITS2 was selected for amplification and recognition with a real‐time PCR assay, based on a combination of three specific motifs, each recognized by a specific fluorescent probe. Similarly, a Scorpion probe was designed for the RT‐PCR quantification of P. c. chlamydosporia. For this purpose, the ITS‐2 rDNA gene of the fungus was sequenced from a number of Italian isolates. A conserved region unique for P. c. chlamydosporia found in the ITS‐2 rDNA gene was used. The probes allowed recognition of single juveniles of M. incognita and of the mycelium‐ or soil‐extracted fungal DNA. The potentialities of the detection procedures are discussed.     

Distribution and characterization of Dothistroma needle blight pathogens on <Emphasis Type="Italic">Pinus mugo</Emphasis> in Slovakia

The occurrence and distribution of Dothistroma needle blight (DNB) on Pinus mugo was studied in 2014–2015 around the Slovakia. In total, 42 localities were investigated both native and planted ones. Symptoms of DNB were observed on 35 localities only on planted shrubs. All these 35 localities are new P. mugo DNB stands. No DNB symptoms were observed in natural and naturally regenerated plantations. DNA was extracted from a total of 236 isolates and eight needle samples. Based on the ITS-rDNA comparisons and using species specific primers, both pathogenic Dothistroma species were detected: D. septosporum and D. pini. Isolates of D. septosporum had ITS sequences identical to D. septosporum from Europe and both mating types were identified with slight predominance of MAT2. The ratio of D. septosporum mating types varies significantly between sites, ranging from an equal proportion of each mating type to single mating type populations. D. pini ITS sequence grouped with D. pini from Ukraine, Russia and Switzerland and only MAT2 was found.     

Wilt and root rot of poinsettia caused by three high-temperature-tolerant Pythium species in ebb-and-flow irrigation systems

Poinsettia plants growing in ebb-and-flow irrigation systems developed wilting and root rot during the summer growing seasons of 2010 in Gifu Prefecture and 2011 in Aichi Prefecture. Pythium species were isolated from roots with rot symptoms. The isolates were identified as P. helicoides and P. myriotylum on the basis of morphological characteristics and sequence homologies in the rDNA internal transcribed spacer regions. In pathogenicity tests, these isolates caused severe wilting and root rot. This is the first report of poinsettia root rot disease caused by P. helicoides and P. myriotylum, although P. aphanidermatum was reported as a pathogen of poinsettia root rot. To better understand these diseases, we performed an epidemiological study of three high-temperature-tolerant Pythium species, P. aphanidermatum, P. helicoides and P. myriotylum. Disease incidence as a percentage of diseased plants was greatest at 35 °C for all three species. Disease severity using the rating scale of root rot was also highest at 35 °C, particularly with high zoospore inoculum densities (100.0 zoospores/mL). Although the disease incidence and severity were reduced at lower temperatures, the three Pythium species were able to cause disease at temperatures as low as 20 °C.