Molecular characterization of <Emphasis Type="Italic">Ditylenchus dipsaci</Emphasis> on Onion in Turkey

Ditylenchus dipsaci is a species complex including diploid and polyploid individuals. The onion race of D. dipsaci is a sensu stricto group and has a wide range of host spectrum. Identification of the D. dipsaci onion race is difficult using morphological and morphometrical methods. Species specific primers are mostly used in molecular approaches for identification of D. dipsaci populations. Fifty one morphologically selected Ditylenchus spp. populations from onion production areas in Turkey were subjected to molecular identification using four D. dipsaci species specific primer sets (PF1-PR1, PF2-PR2, DdpS1-rDNA2, DitNF1- rDNA2, H05-H06) targeting 5.8S and 18S rDNA, ITS1 and flanking ITS regions. Thirty nine percent of the nematode samples were positive with four primers tested, while four of the nematode samples gave specific bands with H05-H06 primers. Ditylenchus dipsaci sensu stricto was identified with specific primer sets in Adana, Hatay, Tekirdag, Bursa, Aksaray, Karaman, Eskisehir and Ankara provinces in Mediterranean, Trace, Aegean and Central Regions in Turkey.     

Ditylenchus gigas n. sp. parasitizing broad bean: a new stem nematode singled out from the Ditylenchus dipsaci species complex using a polyphasic approach with molecular phylogeny

Morphologial, biochemical, molecular and karyological analyses of different populations and races of the stem and bulb nematode Ditylenchus dipsaci have suggested that it represents a species complex, of which only D. dipsaci sensu stricto and its morphologically larger variant, known as the giant race of the stem and bulb nematode, are plant parasites of economic importance. The present study singles out the giant race from this complex, herein described as a new species named Ditylenchus gigas n. sp., on the basis of morphological and molecular data obtained from several populations collected from broad beans in southern Italy, southern Spain and Lebanon. The new species epithet, which refers to the large body size of the nematode with respect to the normal races, must be considered to be conspecific with the D. dipsaci‘giant race’ from Fabaceae in recent literature. Morphologically, the new species is characterized by a body size 1·5–2 times longer than the ‘normal race’, stylet delicate (11·5–13·0 μm long) with knobs distinctly sloping backwards, and long post‐vulval uterine sac (81–150 μm long). Results of molecular analysis of rDNA sequences including the ITS1‐5.8S‐ITS2 region, the D2–D3 fragment of the 28S gene, the small 18S subunit, the partial mitochondrial gene for cytochrome c oxidase I (mtCOI), and hsp90 gene sequences, support the new taxonomic species status for the former D. dipsaci giant race from Vicia faba, and clearly distinguish D. gigas n. sp. from D. dipsaci sensu stricto.     

Molecular Diagnostics, Taxonomy, and Phylogeny of the Stem Nematode Ditylenchus dipsaci Species Complex Based on the Sequences of the Internal Transcribed Spacer-rDNA

ABSTRACT The stem nematode Ditylenchus dipsaci is of great economic importance worldwide as a parasite of agricultural crops and horticultural plants. The internal transcribed spacer (ITS) of rDNA from 23 populations of the D. dipsaci complex from various host plants were amplified and sequenced. Seven previously studied populations were also included in the study. The phylogenetic analysis of the full ITS and ITS2 sequence alignments using minimum evolution, maximum parsimony, and Bayesian inference under the complex model of DNA evolution revealed trees with two main clades: (i) D. dipsaci sensu stricto with diploid chromosome numbers and comprising most isolates from agricultural, ornamental, and several wild plants, and (ii) Ditylenchus spp. with polyploid chromosome numbers, reproductively isolated from diploid populations, and subdivided into six subclades ("giant race" from Vicia faba, Ditylenchus species parasitizing various Asteraceae, and a Ditylenchus sp. from Plantago maritima). Using the energy minimization approach and comparative sequence analysis, it has been found that the secondary structure of ditylenchid ITS2 is organized in three main domains. The importance of knowledge on the RNA structure for phylogenetic analysis is discussed. Conventional polymerase chain reaction (PCR) and real-time PCR with SYBR green dye I with a species specific primer have been developed for detection and quantification of D. dipsaci sensu stricto Validation tests revealed a rather high correlation between real numbers of fourth-stage juveniles of the stem nematodes in a sample and expected numbers detected by real-time PCR. Problems of accuracy of quantification are discussed.     

Host status and reaction of <Emphasis Type="Italic">Medicago truncatula</Emphasis> accessions to infection by three major pathogens of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches.     

PM 7/87 (2) Ditylenchus destructor and Ditylenchus dipsaci

Specific scope

This Standard describes a diagnostic protocol for Ditylenchus destructor and Ditylenchus dipsaci. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology. 2

Specific approval and amendment

Approved in 2008‐09. This revision was prepared on the basis of the IPPC Diagnostic Protocol adopted in 2015 on D. dipsaci and D. destructor (Annex 8 to ISPM 27 Diagnostic protocols for regulated pests). The EPPO Diagnostic Protocol is consistent with the text of the IPPC Standard for morphological identification for this species. For comparison with other species the IPPC table includes Ditylenchus africanus whereas the EPPO table includes Ditylenchus convallariae due to the different distribution of the species. The molecular tests for which there is experience in the EPPO region are described in full in the appendices (some of these are additional tests to those in the IPPC protocol). Reference is given to the IPPC protocol for tests for which there is little experience in the EPPO region. DNA barcoding is also included. Revision approved in 2017‐04.     

A scheme of plant certification for production of nematode-free stocks1

A scheme for the control of plant-parasitic nematodes in the production of planting stocks of garlic (Allium sativum) and strawberry (Fragaria × ananassa) is described. Nematodes controlled are Ditylenchus dipsaci on garlic and D. dipsaci, Aphelenchoides ritzema-bosi, A. fragariae and Meloidogyne spp. on strawberry. The scheme gave very satisfactory results in the protection of garlic cultivations for over 10 years and strawberry plantations for at least 8 years.     

Characterization of two races of the stem and bulb nematode (Ditylenchus dipsaci) in Israel

Two distinct races ofDitylenchus dipsaci in Israel were identified: one, which infects and damages onion and garlic, reproduces on pea, but does not infect phalaris grass; and a second, which infects and damages phalaris—and, probably, also ‘Saia’ oats—but fails to infect onion and garlic. A new ‘garlic’ race of the nematode does not appear to have been introduced into Israel together with the ‘Lavinia’ garlic cultivar, as previously speculated, but rather the introduced Lavinia clone is highly susceptible to the existing ‘onion and garlic’ race ofD. dipsaci.     

The main parasitic nematodes on agricultural crops in Albania and their control1

From studies carried out on soil and root samples from the different agricultural crops in Albania, the following nematode genera have been detected: Meloidogyne, Ditylenchus, Tylenchulus, Globodera, Heterodera, Xiphinema, Pratylenchus, Helicotylenchus, Paratylenchus, Longidorus and Rotylenchus. The commonest species of nematodes are: M. incognita, X. pachtaicum, D. dipsaci, D. destructor, T. semipenetrans, G. rostochiensis, H. schachtii and P. penetrans. Trials on chemical control of root-knot nematodes on tomato have indicated that methyl bromide (500 kg ha^-1) and dichloropropane/dichloropropene are still the best chemicals. Certification of garlic crops, with selection of bulbs before and after sowing, has increased garlic yields to 5–10t ha^-1. Chemical methods to control D. dipsaci are not usually economical.     

The spiculum: an additional useful character for the identification of Ditylenchus dipsaci and D. destructor (Nematoda: Anguinidae)

The quarantine nematodes Ditylenchus dipsaci and D. destructor are often routinely identified by morphology. This study identifies optimal conditions for specimen preparation of samples in order that the spiculum shape is clearly observable without distortion and allows the 2 species to be distinguished.     

Detection of <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> by PCR using novel <Emphasis Type="Italic">virD2</Emphasis> gene-specific primers that discriminate two subgroups

Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA purification kit as a step for the isolation of nucleic acids.     

Simultaneous identification of species and trichothecene chemotypes of <Emphasis Type="Italic">Fusarium asiaticum</Emphasis> and <Emphasis Type="Italic">F. graminearum</Emphasis> sensu stricto by multiplex PCR

A multiplex PCR assay was developed for simultaneous identification of the species and trichothecene chemotypes for Fusarium asiaticum and F. graminearum sensu stricto based on the genes related to trichothecene biosynthesis. PCR was carried out in a single reaction with three pairs of primers designed for the tri6 region and one pair of primers designed for tri3. We confirmed that the multiplex PCR was able to identify species and chemotypes for all tested strains of F. asiaticum and F. graminearum s. str. isolated in Japan. This technique would be a useful and rapid tool for diagnosis, epidemiology, and population structure studies of the F. graminearum complex in Japan.     

<Emphasis Type="Italic">Cercospora zeina</Emphasis> is the causal agent of grey leaf spot disease of maize in southern Africa

The aim of our study was to identify the causal agent of grey leaf spot disease of maize in southern Africa. Single-conidial cultures were recovered from maize leaves with typical disease symptoms sampled from several fields in South Africa, Zambia and Zimbabwe. Morphology, cultural characteristics, and a PCR-based test using Cercospora zeae-maydis and C. zeina-specific primer sets identified all single-conidial cultures as C. zeina. In addition, sequence alignment of DNA fragments of the internal transcribed spacer region (ITS1, ITS2, and the 5.8S gene) and elongation factor 1-α grouped all cultures in the same clade as the C. zeina ex-type culture CBS 118820. To by-pass cultivation of the slow-growing fungus, a rapid method to isolate DNA directly from lesions was successfully applied for PCR identification of C. zeina with species-specific ITS and histone primers. Koch’s postulates were fulfilled for C. zeina by artificially inoculating maize plants in a greenhouse, re-isolating conidia emerging from lesions and verifying pathogen identity with molecular techniques. These results provide evidence that confirms the presence of C. zeina and absence of C. zeae-maydis in commercial maize plantations in southern Africa.     

Genetic relationships among strains of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis>pv. <Emphasis Type="Italic">campestris</Emphasis>revealed by novel rep-PCR primers

Novel primers for rep-PCR were developed with the original software and based on `ancient diverged periodical sequences'. Rep-PCR with these primers was applied to study genetic relationships among 51 Xanthomonas campestris strains. The strains were collected from different countries including Russia, Japan, UK, Germany and Hungary. Reference strains of three X. campestrispathovars and five other Xanthomonas species were included. Based on qualitative differences in amplification profiles, the strains were divided into four major groups. Two subgroups recognised within X. campestrispopulation were similar to RFLP haplotypes. The third subgroup included strains of two other pathovariants and Japanese isolates of X. campestris pv. campestriswhile the fourth group comprised the other species of Xanthomonas. The analysis of the diversity within X. campestris resulted in the conclusion that isolates belong to distinct clonal populations (subgroups). The differences between the subgroups of X. campestris were only slightly smaller than between species of Xanthomonas. A PCR fragment about 600 bp amplified by primer KRPN2 was found in nearly all tested strains of X. campestris.SCAR primers designed for this marker produced a single specific band for strains of X. campestris, but not for other Xanthomonas, Pseudomonas and Erwiniastrains tested. Application of the new primer set for rep-PCR offers a rapid, simple and reproducible method for identification of bacterial strains. The X. campestris-specific SCAR primers may be used in diagnostics of this important plant pathogen.     

Genomic Variation within Monilinia laxa, M. fructigena and M. fructicola, and Application to Species Identification by PCR

Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. fructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isolates collected in France was amplified by Polymerase Chain Reaction (PCR) using universal primers and sequenced. Multiple alignment of the ITS sequences and comparison with published sequences revealed very little intraspecific variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356bp fragment for each of the three species. The specificity of the three primer pairs was successfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countries, unequivocally confirming the identification of each isolate based on morphological and cultural traits. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assays was also successfully confirmed with DNA extracted from different fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run. Moreover, detection and identification of the Monilinia species were successfully achieved directly on diseased fruits. This simple and rapid method can be particularly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.     

Identification of the Beet Cyst Nematode Heterodera schachtii by PCR

PCR-RFLPs of ITS-rDNA and PCR with species-specific primers were developed for identification of cysts and juveniles of the beet cyst nematode Heterodera schachtii. Restrictions of PCR product by MvaI or ScrFI distinguish H. schachtii, H. betae, H. trifolii and H. medicaginis. RFLP profiles with eight restriction enzymes for these four nematode species are presented. Based on Internal Transcribed Spacer sequences of populations from several Schachtii group species, a specific primer for H. schachtii was designed, permitting amplification of the target sequence from juveniles and cysts of the beet cyst nematode. A duplex PCR protocol tested with a wide range of nematode samples is described.     

PCR‐based identification of Pythium spp. causing cavity spot in carrots and sensitive detection in soil samples

On the basis of ITS sequences PCR primers were designed for the identification of the five Pythium species found to be most important for the development of carrot cavity spot in Norway: P. intermedium, P. sulcatum, P. sylvaticum, P. violae and P. ‘vipa’. The P. ‘vipa’ isolates had a unique ITS sequence, differed morphologically from all other Pythium isolates, and thus probably represent a new species. The PCR primers were species‐specific with no cross‐reaction to other Pythium species or to fungal isolates from carrot tested. The detection limits varied for the different primer pairs. The two most sensitive assays allowed detection of as little as 5 fg DNA. All five Pythium species could be detected in lesions from diseased carrots. Weak positive signals were obtained from some carrot samples without symptoms. PCR assays allowed detection of pathogens in soil. In samples of soil known to produce cavity spots on cropped carrots, strong signals were obtained. In several soil samples more than one of the five Pythium species could be detected. The utilization of this diagnostic PCR assay in analysis of field soil and carrot tissue might in the future be exploited to reduce the incidence of this serious carrot disease.     

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。     

Specific Polymerase Chain Reaction Identification of Venturia nashicola Using Internally Transcribed Spacer Region in the Ribosomal DNA

ABSTRACT A technique based on the polymerase chain reaction (PCR) was developed for the identification of Venturia nashicola using nucleotide sequence information of the ribosomal DNA region. The complete internal transcribed spacer (ITS) region of V. nashicola strains and phylo-genetically related species was amplified with the two universal ITS1 and ITS4 primers, sequenced, and digested with five restriction enzymes. The alignment of nucleotide sequences and analyses of digestion patterns indicated constant polymorphisms between V. nashicola and related species at nucleotides 126 and 127, which overlapped a TaqI restriction site. An oligonucleotide primer named A126 was designed for identifying this variable region. A primer set (A126 and ITS4) that allowed the amplification of a 391-bp DNA fragment within the ITS region by PCR was specific to V. nashicola when it was checked against fungal genomic DNAs of related fungi. This primer set was a good candidate for a species-specific reagent in a procedure for identification of V. nashicola by PCR.     

Phylogenetic Analysis of Sugarcane Rusts Based on Sequences of ITS, 5.8 S rDNA and D1/D2 Regions of LSU rDNA

Phylogenetic analysis of sugarcane rusts based on sequences of ITS and the 5.8 S rDNA revealed two highly divergent ITS groups among isolates of Puccinia sp. sensu Muta, 1987 and P. kuehnii specimens. Although there is sufficient divergence (exceeding normal intraspecific variation) between the ITS regions of the two groups to support separation into different species, unusually high homology of the ITS group I sequences with those of members of Cronartium and identical sequences of the D1/D2 regions of the LSU rDNA for all the isolates of “Puccinia sp.” and P. kuehnii that otherwise exhibited different ITS sequences, suggest that the two highly divergent sequences may have resulted from abnormal genetic events leading to non-orthologous, intraspeciflc polymorphisms. The other sugarcane rust, P. melanocephala and the grass rusts, P. miscanthi and P. rufipes, were separated from “Puccinia sp.” and P. kuehnii and from each other in D1/D2 region analyses, indicating that D1/D2 region sequences may more correctly reflect phylogenetic relationships in these rusts than do the ITS regions. Further studies to examine differences in patho-genicity or finer morphological features within P. kuehnii that may be correlated with the high divergence in ITS sequences and experiments to determine if these two sequence types represent intraspeciflc polymorphism are necessary. Received 11 October 2000/ Accepted in revised form 24 November 2000