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 <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.     

A quantitative real‐time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds

Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum lindemuthianum. Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real‐time PCR‐based diagnostic assay was developed for detecting C. lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA (rDNA) region consisting of part of the18S rDNA, 5^.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C. lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C. lindemuthianum genomic DNA. To explore the correlation between the lesion area and the DNA amount of C. lindemuthianum in bean seed, seeds of the navy bean cultivar Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays.     

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.     

Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab‐on‐a‐chip DNA hybridization array

A novel DNA‐chip hybridization assay that uses the ras‐related GTP‐binding protein 1 gene (Ypt1) was developed for the identification of several devastating Phytophthora species. The hybridization was conducted in a portable microfluidic lab‐on‐a‐chip device for fast and accurate detection of 40 Phytophthora, two Pythium and one Phytopythium species. Moreover, the functionality of the Ypt1 region was examined in comparison to an array for the internal transcribed spacer (ITS) region by in silico modelling. The difference in species‐specific capture probe sequences was lower for the ITS than for the Ypt1 region. While ITS‐probes of Phytophthora ramorum, Phytophthora fragariae and Phytophthora lateralis cross‐reacted with up to 11 non‐target species, Ypt1‐probes were specific except for P. fragariae/Phytophthora rubi. First analyses of artificially inoculated Rhododendron leaves successfully demonstrated the usability of the respective capture probes for the Ypt1 and the ras‐related plant protein Rab1a gene region. The on‐chip hybridization enabled the detection of up to 1 pg μL^?1 target DNA depending on the species examined. Due to the complementarity of ITS and Ypt1 genetic features, the use of multiple loci is recommended to identify targets of different taxonomic rank.     

甘肃定西地区马铃薯线虫病病原的分离鉴定

为明确甘肃省定西市马铃薯线虫病的病原种群分类地位,采用形态学结合分子生物学的方法对该地区马铃薯上的4个线虫群体进行了鉴定,观察和测量其形态特征值,基于r DNA-ITS序列以UPGMA法构建线虫群体的系统发育树,并按照柯赫氏法则进行了致病性测定。结果表明,4个线虫群体在形态学上与马铃薯腐烂茎线虫Ditylenchus destructor一致,但群体DX27与群体DX11、DX16、DX19雌虫的体长、体长/食道长、体长/尾长值存在极显著差异。利用通用引物TW81/AB28扩增r DNA-ITS序列均获得长度为915 bp的片段;序列比对分析表明,群体DX27与其它3个群体相比在ITS1区的第96~255 bp片段内有25个碱基的差异;系统发育树显示,群体DX27与C型群体聚为1支,群体DX11、DX16、DX19与B型群体聚为1支。根据形态学特征及r DNA-ITS序列分析结果确定该病原线虫为马铃薯腐烂茎线虫,其中群体DX27属于C型,群体DX11、DX16、DX19属于B型。     

Occurrence and ITS diversity of wood‐associated Bursaphelenchus nematodes in Scots pine forests in Switzerland

The quarantine pathogen Bursaphelenchus xylophilus (pine wood nematode, PWN) represents a serious threat for Pinus species in Europe. To exclude its presence in Switzerland, in 2010 and 2011 a countrywide survey was conducted in 102 Pinus sylvestris stands, chosen according to whether they contained dying or dead trees or were located in areas at risk of PWN introduction. In total, 285 trees (1–5 per site) were sampled. Nematodes were extracted from wood chips using a standard procedure, and identified to species by internal transcribed spacer (ITS) sequencing. Bursaphelenchus species were present in 34% of the trees, but no B. xylophilus was identified, i.e. PWN is still not present in Switzerland. The nematodes found belonged to seven different species, with B. vallesianus the most frequent species, followed by B. sexdentati, B. mucronatus kolymensis and B. eggersi. Three other species (B. borealis, B. pinophilus, B. poligraphi) were each only present in one or two trees. Three groups of sequences could not be assigned to a species because of the lack of matching reference sequences. The species composition found in Switzerland suggests co‐existence of southern and central European Bursaphelenchus species. Intraspecific ITS variability differed considerably among the four most common species. Bursaphelenchus eggersi, B. mucronatus kolymensis and B. sexdentati had several variable sites in the ITS region, resulting in multiple ITS genotypes in each species. In contrast, all 99 B. vallesianus isolates had an identical ITS region. This could indicate a founder effect, and possibly that B. vallesianus is not native to the Alpine region.     

Characterization of Botryosphaeriaceae from plantation‐grown Eucalyptus species in South China

The Botryosphaeriaceae is a species‐rich family that includes pathogens of a wide variety of trees, including Eucalyptus species. Symptoms typical of infection by the Botryosphaeriaceae have recently been observed in Eucalyptus plantations in South China. The aim of this study was to identify the Botryosphaeriaceae associated with these symptoms. Isolates were collected from branch cankers and senescent twigs of different Eucalyptus spp. All isolates resembling Botryosphaeriaceae were separated into groups based on conidial morphology. Initial identifications were made using PCR‐RFLP fingerprinting, by digesting the ITS region of the rDNA operon with the restriction enzymes CfoI and KspI. Furthermore, to distinguish isolates in the Neofusicoccum parvum/N. ribis complex, a locus (BotF15) previously shown to define these species, was amplified and restricted with CfoI. Selected isolates were then identified using comparisons of DNA sequence data for the ITS rDNA and translation elongation factor 1‐alpha (TEF‐1α) gene regions. Based on anamorph morphology and DNA sequence comparisons, five species were identified: Lasiodiplodia pseudotheobromae, L. theobromae, Neofusicoccum parvum, N. ribis sensu lato and one undescribed taxon, for which the name Fusicoccum fabicercianum sp. nov. is provided. Isolates of all species gave rise to lesions on the stems of an E. grandis clone in a glasshouse inoculation trial and on the stems of five Eucalyptus genotypes inoculated in the field, where L. pseudotheobromae and L. theobromae were most pathogenic. The five Eucalyptus genotypes differed in their susceptibility to the Botryosphaeriaceae species suggesting that breeding and selection offers opportunity for disease avoidance in the future.     

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.     

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.     

Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index,X. diversicaudatum,X. vuittenezi and X. italiae,and for the quantification of X. index numbers

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.     

A nested‐PCR method for rapid detection of Sclerotinia sclerotiorum on petals of oilseed rape (Brassica napus)

This study established a quick and accurate method to detect petal infection of oilseed rape (Brassica napus) by Sclerotinia sclerotiorum using a nested‐PCR technique. DNA samples were extracted from each petal using a microwave method, followed by two rounds of PCR amplification. The first‐round PCR amplification was performed using the universal fungal primer pair ITS4/ITS5, and the second‐round amplification with a specific primer pair XJJ21/XJJ222, which was designed using the single‐nucleotide polymorphisms among nuclear rDNA ITS sequences of Sclerotinia spp., Botrytis spp. and other selected fungi. The established technique is rapid and inexpensive, and has a high degree of specificity and sensitivity. This assay can distinguish Sclerotinia spp. from other fungi, including Botrytis cinerea, a closely related and frequent cohabitant on oilseed rape petals, and can detect 50 fg genomic DNA, five ascospores of S. sclerotiorumin vitro or 50 ascospores of S. sclerotiorum on one petal in approximately 6 h, even in the presence of a high background of oilseed rape DNA. This technique was successfully applied in detecting natural petal infections.     

Two novel and potentially endemic species of Phytophthora associated with episodic dieback of Kwongan vegetation in the south‐west of Western Australia

Two novel homothallic species of Phytophthora causing dieback of Kwongan vegetation in south‐west Western Australia are described here as Phytophthora arenaria sp. nov. and Phytophthora constricta sp. nov. DNA sequencing of the ITS rDNA and cox1 gene confirmed that P. arenaria and P. constricta are unique species residing in ITS clades 4 and 9, respectively. Phytophthora arenaria has been isolated from vegetation occurring on the northern sandplains which are warmer and drier than the southern sandplains from which P. constricta has been predominantly isolated, and both species appear morphologically and physiologically well adapted to the ecosystems in which they occur. Both species have been associated mainly with dead and dying Banksia species and the pathogenicity of both P. arenaria and P. constricta to Banksia attenuata was confirmed in this study. The combination of unique DNA sequences, including considerable variation in cox1 sequence data, thick oospore walls and physiological characteristics that appear to be adaptations favouring survival in the harsh Kwongan ecosystem suggest that these species may be endemic to Western Australia.     

溴氰菊酯对马铃薯腐烂茎线虫的毒力及对其运动和摄食的影响

采用药液浸渍法、沙柱法以及与荧光染料Cy3共孵育的方法,以丙溴磷、克百威和阿维菌素为对照药剂,测定了溴氰菊酯对马铃薯腐烂茎线虫Ditylenchus destructor的毒力以及对其运动扩散和摄食的影响。结果表明:溴氰菊酯对马铃薯腐烂茎线虫具有一定的杀灭活性,其LC50值为459.8mg/L,活性低于丙溴磷(159.9mg/L)、克百威(331.9mg/L)和阿维菌素(257.3mg/L);溴氰菊酯对马铃薯腐烂茎线虫运动扩散的抑制作用IC50值为3.1mg/L,其活性低于阿维菌素(0.8mg/L),但高于丙溴磷(8.3mg/L)和克百威(16.1mg/L)。当丙溴磷和克百威质量浓度分别低至0.6和40mg/L时,可刺激90%以上的线虫摄食;当丙溴磷和克百威质量浓度最低分别为360和300mg/L时,可抑制全部线虫的摄食;溴氰菊酯与阿维菌素则对线虫的摄食无刺激作用;用10mg/L的丙溴磷处理线虫2h后再分别用阿维菌素和溴氰菊酯处理,发现阿维菌素和溴氰菊酯质量浓度最低分别为20和200mg/L时可抑制全部马铃薯腐烂茎线虫的摄食。研究表明,溴氰菊酯对马铃薯腐烂茎线虫具有较高的活性,其在线虫防治领域的开发应用潜力较好。     

Note: Molecular identification of three entomopathogenic nematodes from turkey by PCR-RFLP of the ITS regions

Molecular identification methods are widely used for the classification of organisms worldwide. Entomopathogenic nematodes are the most often isolated insect parasitic nematodes in the tropical and subtropical regions. In our investigation, PCR-RFLP (Polymerase Chain Reaction — Restriction Fragment Length Polymorphism) of the ITS region (Internal Transcribed Spacer) on the ribosomal (r) DNA of three entomopathogenic nematodes isolated from Ankara, Turkey, was analyzed for identification. The ITS region of rDNA was amplified by PCR and then digested with the following nine restriction enzymes: Alu I, Dde I, Hae III, Hha I, Hind III, Hinf I, Hpa II, Rsa I and Sau 3AI. The amplified and restricted sequences of the ITS regions were separated by agarose gel electrophoresis and the RFLP patterns of these three species were shown in this study. According to our results, these species were identified asSteinernema feltiae, Steinernema carpocapsae andHeterorhabditis bacteriophora. http://www.phytoparasitica.org posting Nov. 4, 2005.     

Molecular analysis of Phytophthora diversity in nursery‐grown ornamental and fruit plants

The genetic diversity of Phytophthora spp. was investigated in potted ornamental and fruit tree species. A metabarcoding approach was used, based on a semi‐nested PCR with Phytophthora genus‐specific primers targeting the ITS1 region of the rDNA. More than 50 ITS1 sequence types representing at least 15 distinct Phytophthora taxa were detected. Nine had ITS sequences that grouped them in defined taxonomic groups (P. nicotianae, P. citrophthora, P. meadii, P. taxon Pgchlamydo, P. cinnamomi, P. parvispora, P. cambivora, P. niederhauserii and P. lateralis) whereas three phylotypes were associated to two or more taxa (P. citricola taxon E or III; P. pseudosyringae, P. ilicis or P. nemorosa; and P. cryptogea, P. erythroseptica, P. himalayensis or P. sp. ‘kelmania’) that can be challenging to resolve with ITS1 sequences alone. Three additional phylotypes were considered as representatives of novel Phytophthora taxa and defined as P. meadii‐like, P. cinnamomi‐like and P. niederhauserii‐like. Furthermore, the analyses highlighted a very complex assemblage of Phytophthora taxa in ornamental nurseries within a limited geographic area and provided some indications of structure amongst populations of P. nicotianae (the most prevalent taxon) and other taxa. Data revealed new host–pathogen combinations, evidence of new species previously unreported in Italy (P. lateralis) or Europe (P. meadii) and phylotypes representative of species that remain to be taxonomically defined. Furthermore, the results reinforced the primary role of plant nurseries in favouring the introduction, dissemination and evolution of Phytophthora species.     

Quantification of Mirafiori lettuce big‐vein virus and its vector,Olpidium virulentus,from soil using real‐time PCR

Big vein disease of lettuce (Lactuca sativa) is an economically important disease transmitted through soil by Olpidium virulentus, and has occurred in most production areas worldwide. The disease is assumed to be caused by Mirafiori lettuce big‐vein virus (MiLBVV). To understand the dynamics of the virus and its vector, MiLBVV and O. virulentus were directly detected in soil. DNA and RNA were extracted from 5 g soil using a bead beating method, followed by purification using adsorption to a column. Detection and quantification were performed using real‐time PCR and a TaqMan probe that was prepared based on the CP region of MiLBVV and the rDNA‐ITS region of O. virulentus, respectively. Furthermore, using a visual assessment of the incidence rate of big vein disease on lettuce in agricultural fields, the C_t values of MiLBVV and O. virulentus from soil were also determined using real‐time PCR. The results showed that MiLBVV concentrations in the soil were high in the field, as also determined by a visual assessment of the incidence rate of big vein disease on lettuce. However, the amount of O. virulentus in soil was not directly correlated with the incidence of MiLBVV. From these results, it is suggested that the risk of lettuce crops developing big vein disease can be estimated using an index of the amount of MiLBVV in the soil.     

Genetic relationship between Echinochloa crus‐galli and Echinochloa oryzicola accessions inferred from internal transcribed spacer and chloroplast DNA sequences

Barnyardgrass, hexaploid Echinochloa crus‐galli, is considered to arise from the hybridization between tetraploid Echinochloa oryzicola and an unknown diploid species. The genetic relationship between E. crus‐galli and E. oryzicola was examined to investigate the position of E. oryzicola in the evolutionary process of E. crus‐galli, based on the nuclear DNA internal transcribed spacer (ITS) and the chloroplast cpDNA trnT‐L, trnL intron, and trnL‐F regions. New World E. crus‐galli was clearly separated from Eurasian E. crus‐galli and showed a close relationship to the American taxa, Echinochloa crus‐pavonis and Echinochloa walteri, in both the ITS and chloroplast DNA. The nrDNA ITS sequences indicated no differentiation between the Eurasian E. crus‐galli and E. oryzicola, in contrast to their clear divergence in the cpDNA sequence. The present results suggest that E. oryzicola is the male donor of E. crus‐galli.     

15种植物源化合物对马铃薯茎线虫的活性比较

利用离体生测方法,系统测定和比较了15种植物源化合物对马铃薯茎线虫的活性。结果表明,dl-薄荷醇和香茅醇等10种化合物对马铃薯茎线虫具有较高的活性;选用6种高活性化合物对马铃薯茎线虫的毒力测定结果表明:处理24 h和48 h后6种化合物的LC₅₀由小到大的顺序分别为:油酸<间苯三酚<间苯二酚<柠檬酸<dl-薄荷醇;油酸<间苯二酚<香茅醇<间苯三酚<柠檬酸<dl-薄荷醇。