Vulval Cone Research Articles

First Report of Cyst Nematode (Heterodera iri) in Ohio

HomePlant DiseaseVol. 102, No. 5First Report of Cyst Nematode (Heterodera iri) in Ohio PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Cyst Nematode (Heterodera iri) in OhioS. Joseph, F. Akyazi, A. Habtewld, T. Mekete, T. Creswell, G. E. Ruhl, and J. FaghihiS. JosephSearch for more papers by this author, F. AkyaziSearch for more papers by this author, A. HabtewldSearch for more papers by this author, T. MeketeSearch for more papers by this author, T. Creswell†Corresponding author: T. Creswell; E-mail: E-mail Address: creswell@purdue.eduSearch for more papers by this author, G. E. RuhlSearch for more papers by this author, and J. FaghihiSearch for more papers by this authorAffiliationsAuthors and Affiliations S. Joseph F. Akyazi A. Habtewld T. Mekete , Entomology and Nematology Department, University of Florida, Gainesville, 32611 T. Creswell † G. E. Ruhl , Plant and Pest Diagnostic Laboratory, Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 J. Faghihi , Department of Entomology, Purdue University, West Lafayette, IN 47907. Published Online:23 Mar 2018https://doi.org/10.1094/PDIS-11-17-1846-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat The cyst nematodes (Heterodera spp.) are a major group of plant-parasitic nematodes and of great economic importance in many countries throughout the world, causing considerable yield losses to many important crops (Jones et al. 2013). The cereal cyst nematodes (CCNs) are a widespread and important group of cyst nematodes that restrict production of cereal crops throughout the world (Rivoal and Cook 1993). The CCNs that belong to the Heterodera avenae complex infect roots of cereals and grasses (Handoo 2002). Four species from this group have been reported in the United States, including H. avenae, H. filipjevi, H. mani, and H. ustinovi (syn. H. iri). H. iri has previously been reported only in New York, Michigan, Connecticut, Massachusetts, and Rhode Island (LaMondia and Wick 1992). Cyst nematodes were identified from an Ohio bentgrass golf course sample submitted to the Purdue Plant and Pest Diagnostic Lab and the Purdue Nematology Lab for diagnosis in 2016. The affected turf had irregular patches of thinning, translucent leaves, and reduced root systems. Cysts were collected from the infested soil by the modified Jenkins sugar flotation method (Jenkins 1964) and identified by morphological and molecular study. Morphometrics (mean ± SD) of second-stage juveniles (n = 12): body length = 661.7 ± 23.8 µm; a = 29.6 ± 1.3 µm; c = 7.3 ± 0.3 µm; c′ = 5.2 ± 0.5 µm; stylet length = 27.3 ± 0.5 µm; maximum body width = 22.4 ± 0.4 µm; body width at anus = 17.5 ± 2.1 µm; tail length = 90.9 ± 5.1 µm; hyaline tail length = 55.4 ± 4.8 µm; and hyaline tail /stylet length = 2.1 ± 0.2 µm. Morphometrics of cysts (n = 10): length (without neck) = 654.4 ± 82.9 µm; width = 461.8 ± 84.2 µm; neck length = 127.4 ± 20.8 µm; length/width = 1.4 ± 0.2. The vulval cone was bifenestrate with a strong vulval bridge and conspicuous bullae. Morphometrics of vulval cone (n = 7): fenestral length = 42.4 ± 4.1 µm; fenestral width = 24.4 ± 2.1 µm; vulval slit = 12.9 ± 1.4 µm; and vulval bridge = 3.1 ± 0.5 µm. Eggs (n = 10) measured length = 136.5 ± 8.9 µm; width = 43.7 ± 2.8 µm; and length/width = 3.1 ± 0.3. These morphological measurements were highly similar with those for H. iri as described by Mathews (1971). Genomic DNA was extracted from five nematodes. The ribosomal region spanning the internal transcribed spacer (ITS) 1, 5.8S gene, and ITS2 was amplified with forward primers AB28F (5′-GTTTCCGTAGGTGAACCTGC-3′) and reverse primer TW81R (5′-ATATGCTTAAGTTCAGCGGGT-3′). A 973-bp fragment was amplified, and the polymerase chain reaction product was purified and sequenced. The sequence was deposited in GenBank (accession no. KX611142). BLAST search results revealed a sequence similarity of 99% with H. iri (e.g., GenBank accession nos. AY148407.1 and AF274400.1) over 100% query cover. Morphological and molecular identification demonstrated that the population of cyst nematodes from Ohio was H. iri. Reproduction on bentgrass was demonstrated by inoculating 1,000 viable eggs onto 5-week-old bentgrass seedlings grown in 4-in. pots (n = 2) of sterilized sand at 23°C. A pot of noninoculated seedlings served as a control. Sixty days after inoculation, cysts were harvested from the plants, and extraction produced approximately 2,000 eggs, demonstrating multiplication on this host. No cysts were found on the control plants. To our knowledge, this detection represents a new record of H. iri in Ohio.

First Report of the Yellow Nutsedge Cyst Nematode, Heterodera cyperi, in Georgia, U.S.A.

Soil samples collected during a survey for plant-parasitic nematodes in Tift County GA in summer 2017 were submitted for routine diagnosis of nematodes to the Extension Nematology Lab at the Department of Plant Pathology, University of Georgia, Athens, Georgia. Cyst nematodes recovered by centrifugal flotation technique were discovered in the samples from two research sites in a field with a history of tobacco and vegetable production. Cyst nematodes from tobacco (10 cysts/100 cm 3 of soil) and vegetable (2 cysts/100 cm 3 of soil) sites had similar morphological features. Morphology and morphometric measurements of the cysts and J2 ( Fig. 1A-C ) were in agreement with those of Heterodera cyperi ( Golden et al., 1962 ; Romero and López-Llorca, 1996 ). Measurements of J2 ( n = 12) included the length (range = 443-494 μm, mean = 467.4 μm) and width (18.3-24.4 μm, 20.6 μm) of body, stylet (19.1-20.8 μm, 20.3 μm), tail (61.6.0-66.4 μm, 64.2 μm), body width at anus (11.9-14.1 μm, 12.8 μm), and hyaline tail terminus (22.7-29.2 μm, 26.3 μm). The lateral field of J2 had three lines. Cysts ( n = 10; Fig. 1C ) were lemon-shaped, light to dark brown in color with protruding neck and vulval cone. The cysts had ambifenestrated vulval cone and no bullae was present. Morphometrics included body length excluding neck (370.5-714.4 μm, 555.7 μm); body width (165.6-411.1 μm, 310.9 μm); neck length (36.5-66.3 μm, 49.8 μm); fenestra length (26.3-42.5 μm, 35.8 μm), and fenestra width (19.1-31.5 μm, 23.8 μm). DNA was extracted from single cysts ( n = 3) and internal transcribed spacer (ITS) of rRNA and partial cytochrome oxidase I ( COI ) genes were amplified with primers TW81/AB28 and Het-coxiF/Het-coxiR, respectively ( Subbotin et al., 2001 ; Subbotin, 2015 ) and sequenced. The resulting sequences were deposited into the GenBank database (Accession no. MG825344 and MG857126) and also subjected to BLAST searches in the database. ITS sequence of H. cyperi showed 100% similarity (100% coverage) with that of a H. cyperi population from Spain (AF274388). COI sequence of H. cyperi showed 89% similarity (98% coverage) with that of H. guangdongensis (MF425735), and 88% similarity (83% coverage) with that of H. elachista (KC618473). The pathogenicity of H. cyperi was examined under greenhouse conditions using tobacco cv. K340, tomato cv. Tribute, cucumber cv. Thunder, and yellow nutsedge ( Cyperus esculentus L.). 3-wk-old seedlings of the test plants were transferred into Deepot D25L cell containers (5-cm-diam. × 25.4-cm deep) filled with sterilized sand: sand: soil mixture (1:2) and then inoculated with 1,000 eggs and J2 of H. cyperi . The plants were grown for 90 d in a greenhouse before examination of roots and extraction of cysts from the soil. Results showed that the nematode failed to reproduce on tobacco, tomato, and cucumber whereas white females and mature cysts of H. cyperi were observed on yellow nutsedge roots ( Fig. 1E ). The results confirmed that yellow nutsedeg was a host for the nematode, and tobacco, tomato, or cucumber were non-hosts. In the United States, H. cyperi was reported from Florida, North Carolina, and Arkansas ( Subbotin et al., 2010 ) infecting Cyperus spp. Yellow nutsedge is considered a serious weed problem in many cropping systems including peanut, cotton, tobacco, and vegetable crops in the Southern United States. To our knowledge, this is the first report of H. cyperi infecting yellow nutsedge in Georgia. Figure 1Photomicrographs of Heterodera cyperi from yellow nutsedge in Georgia. Whole body (A), the anterior region (B), and the posterior region (C) of J2. Cysts (D) recovered from the soil and the vulval cone of cyst with the ambifenestrate fenestra (E). A mature cyst (F) on the surface of yellow nutsedge root infected with the nematode.

First Report of the Fig Cyst Nematode, Heterodera fici Kirjanova, on Fig Tree, Ficus carica, in Ontario, Canada.

Although fig trees are a popular ornamental fruit tree in subtropical regions, some hardy species, such as Ficus carica, have been grown in the west coast of British Columbia and southern Ontario in Canada. The fig cyst nematode, Heterodera fici Kirjanova, is a pest on fig plants, and the heavy infestation can cause retarded growth and yellowing of leaves (Maqbool et al., 1987). In the spring of 2016, a sample of rhizosphere from a potted fig (F. carica) seedling was submitted to the Nematology Laboratory, Canadian Food Inspection Agency. The sample was collected from a nursery in Niagara-on-the-Lake, Ontario, Canada, during an inspection to support export certification. The fig trees in the nursery had been grown in the outside fields during the growing seasons and potted and moved to indoor during the winters for last 3 years. The sample was subjected to a nematode extraction process, including decanting and sieving and misting, and lemon-shaped cysts and second-stage juveniles of Heterodera sp. were recovered from the sample examined. The morphological and molecular analyses of the cysts, vulval cone, and second-stage juveniles from both the roots and the crushed cysts identified the species as Heterodera fici Kirjanova. The cysts were characterized by their dark brown color and lemon shape, as well as distinct necks and vulval cones. The vulval cones were observed having an ambifenestrate fenestra (Fig. 1AFig. 1Photomicrographs of Heterodera fici on fig tree from Ontario, Canada. A, B. Cyst vulval cones with the ambifenestrate fenestra in A) and well-developed underbridge and bullae in B). C-E. The second-stage juveniles from a crushed cyst with the whole body in C), the anterior region in D) and the posterior region in E).), dome-shaped bullae scattered around the underbridge plane (Fig. 1B), well-developed underbridge (Fig. 1B), and coarse zig-zag ridges surrounding the fenestra on the surface. The cyst measurements (n = 3) were length 608.7 ± 91.6 (506-682) µm, width = 395.3 ± 40.9 (366-442) µm, and length/width ratio = 1.3 ± 0.2 (1.3-1.7) µm. The morphometric characters of the vulval cone were fenestral length = 57.3 ± 2.9 (54-59) µm, fenestral width = 35.3 ± 4 (33-40) µm, and vulval slit = 50.7 ± 1.2 (50-52) µm. Second-stage juveniles tapering posteriorly (Fig. 1C). Stylet well developed, basal knobs rounded, directed slightly anteriorly (Fig. 1D). Tail tapering, with hyaline terminal about half of tail length (Fig. 1E). Second-stage juveniles (n = 16) showed the following morphometric characters: body length = 440.9 ± 20 (408-470) µm, a = 20.3 ± 2.3 (16.9-24), b = 2.7 ± 0.3 (2.3-3.3), c = 8.1 ± 0.6 (7.3-9.3), c' = 3.8 ± 0.3 (3.1-4.3), stylet length = 23.2 ± 0.4 (23-24) µm, anterior end to median bulb = 71.8 ± 4.5 (65-79) µm; maximum body width = 21.9 ± 2 (17-25) µm; body width at anus = 14.4 ± 1.5 (12-17) µm, tail length = 54.6 ± 2.9 (50-60) µm, and hyaline part of tail = 23.7 ± 2.9 (19-28) µm. These observations conform to the published descriptions of Heterodera fici Kirjanova (Kirjanova, 1954; Golden et al., 1988). The slides of the cyst vulval cone and juveniles were deposited in the Canadian National collection of Insects, Arachnids, and Nematodes (Accession no. 14851 to 14853 for the second stage juveniles and 14854-14855 for the cyst cones). For molecular analysis, DNA was extracted from individual juvenile (n = 4) from different cysts. A 1,151-bp fragment of ribosomal DNA containing ITS1-5.8S-ITS2 region was amplified and sequenced using primers 18S (5'-TTGATTACGTCCCTGCCCTTT-3') and 26S (5'- TTTCACTCGCCGTTACTAAGG-3') (Vrain et al., 1992). The sequence was deposited into the GenBank database (Accession no. KY635986 and KY635987) and was compared with published sequences by means of BLAST search in the database (November 2016). The comparison revealed 99.0% to 100% similarity to the sequences of the same genomic region of H. fici from Iran (AF498385) and Georgia (AF274409). Subbotin et al. (2010) summarized the known occurrence of the fig cyst nematode from Belarus, Belgium, Estonia, France, Germany, Greece, Hungary, Italy, The Netherlands, Norway, Poland, Portugal, Russia, Spain, Yugoslavia, China, Georgia, Iran, Turkey, Uzbekistan, Australia, New Zealand, United States (California, Florida, Louisiana, Maryland, and Virginia), Brazil, Algeria, and South Africa. It is an exotic pest to Canada. To our knowledge, this is the first evidence of the occurrence of H. fici in Canada.

First Report of the Cereal Cyst Nematode Heterodera filipjevi on Winter Wheat in Montana

Among the cereal cyst nematode complex, three species-Heterodera avenae, H. filipjevi and H. latipons-are the most destructive for wheat. Although H. avenae occurs in several U.S. states, H. filipjevi was discovered in the United States in Oregon in 2008 and has since been reported only in Washington. In Chouteau County, Montana, a winter wheat field reported to contain H. avenae was observed with localized areas of heavily stunted Yellowstone winter wheat. Roots from affected plants displayed moderate numbers of nematode cysts with no associated proliferation of lateral roots. Living nematode cysts from wheat roots from the affected field were examined morphologically and by PCR for species identification. Observations of morphological characters critical for identification indicated that the specimens were H. filipjevi. Diagnostic measurements of second-stage juveniles included length of body, stylet with anchor-shaped basal knobs, tail, and hyaline tail terminus. The lateral field had four lines; the inner two were more distinct. Shapes of the tail, tail terminus, and stylet knobs were also consistent with H. filipjevi. The lemon-shaped cysts were light brown to yellow and had a zigzag pattern and bifenestrate vulval cone; diagnostic morphometrics included body length excluding neck, body width, length/width, neck length and width, fenestra length and width, heavy underbridge, vulval slit, and many bullae. Molecular analysis of internal transcribed spacer (ITS1 and 2) and 28S rRNA confirmed identity as H. filipjevi. Amplicons generated from three J2 were cloned and sequenced. Sequences from the 28S region were >99.9% identical to those of H. filipjevi from China but distinct from several populations of H. avenae (99.0-99.2%). The ITS rDNA from this population matched with 99.9% identity several H. filipjevi sequences available from GenBank, including ones from Russia, Tajikistan, UK, USA, and China. The next closest species match was H. avenae, showing 96-97% identity to the Montana population. This is the first report of H. filipjevi from Montana. Importantly, eggs from H. filipjevi are noted for hatching in the fall during winter wheat planting and therefore may more greatly affect winter wheat than H. avenae, whose eggs hatch only in the spring after a cold period. Moreover, resistance in wheat against these two nematode species is different and will require additional considerations.

Morphological and morphometrical analysis of Heterodera spp. populations in Jordan

Phenotypic diversity of five Jordanian populations of cyst nematodes, Heterodera spp. collected from five regions from Jordan (Ar-Ramtha, Madaba, Dana, Al-Karak, and Jerash) was investigated. Soil samples were collected from one representative field in each region. Morphological and morphometrical characteristics revealed that Heterodera latipons is dominated in cereal fields at Ar-Ramtha, Madaba, Dana and Al-Karak regions and Heterodera schachtii in Jerash. Cysts populations from all cereal fields had bifenestrate vulval cone and a strong underbridge. Wherever, cysts of the cabbage population had ambifenestrate vulval cone with long vulval slit. The bullae were absent in Ar-Ramtha, Madaba and Dana populations, but present in Al-Karak and Jerash. Based on 12 morphometrical characters, the first three functions in canonical discriminant analysis accounted 99.3% of the total variation. Distance from dorsal gland duct opening to stylet base, underbridge length, a=L/W (body length/midbody width) and length of hyaline tail tip had strong and significant contributions in the first function. While the second function was strongly influenced by length of hyaline tail, fenestral length, fenestral width and tail length. However, the third canonical discriminate function was found to be influenced by stylet length, fenestral length, a=L/W (body length/midbody width) and underbridge width. The graphical representation of the distribution of the samples showed that the first canonical discriminant function clearly separated H. schachtii from Jerash from other populations. Whereas, H. latipons collected from Madaba and Dana were clearly separated in the second function. The results indicated that differences at morphological and morphometrical levels revealed diverse populations of Heterodera spp. in Jordan.

Characterisation of cereal cyst nematodes in Egypt based on morphometrics, RFLP and rDNA-ITS sequence analyses

Morphological and molecular diversity among populations of cereal cyst nematodes (CCN) from wheat production areas in Ismailia province, Egypt, was investigated using light microscopy, ITS-RFLP and sequencing of the rDNA-ITS. CCN were found in five out of seven regions in Ismailia, the highest incidence being found in El Shark (West Sinai). The Egyptian populations were identified as H. avenae according to morphometrics of cyst vulval cone and second-stage juveniles. No differences in ITS-RFLP patterns generated by 17 restriction enzymes were detected among the Egyptian populations although the Egyptian populations could be distinguished from German populations of H. avenae and H. filipjevi. The analyses of ITS region sequences confirmed the species identification of the Egyptian populations as they clustered with H. avenae populations from Iran, Saudi Arabia, India, Israel and China.

Heterodera guangdongensis n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China--a new cyst nematode in the Cyperi group.

Heterodera guangdongensis n. sp. is described from bamboo (Phyllostachys pubescens Mazel) based on morphology and molecular analyses of rRNA D2D3 expansion domains of large subunit (LSU D2D3) and internal transcribed spacer (ITS) sequences. This new species can be classified in the Cyperi group. Cysts are characterized by a prominent, ambifenestrate vulval cone with weak underbridge, a vulva-anus distance of 28.9-35.9 μm and a vulval slit of 31.1-41.0 μm, but without bullae. Females are characterized by a 25.1-27.6 μm stylet with rounded knobs sloping slightly posteriorly. Males are characterized by a 21.5-23.0 μm stylet with knobs slightly projecting or flat anteriorly, lateral field with four lines, and a 22.0-26.0 μm spicule with bifurcate tip. Second-stage juveniles are characterized by a 19.3-21.3 stylet with slightly projecting or anteriorly flattened knobs, lateral field with three lines, a 41.7-61.3 μm tail with finely rounded terminus and hyaline portion forming 43.0-57.1% of the tail length. Molecular analyses show that the species has unique D2D3 and ITS rRNA sequences and RFLP-ITS-rRNA profiles.

Heterodera fengi n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China--a new cyst nematode in the Cyperi group.

Heteroderafengi n. sp. is described and illustrated from bamboo (Phyllostachys pubescens Mazel) based on morphology and molecular analyses of rRNA LSU D2D3 region and ITS. This new species belongs to the Cyperi group. Cysts are characterized by prominent vulval cone with ambifenestrate, bifurcate underbridge that is thicker in middle and a 47.0 (40.0-60.0) µm long vulval slit, but without bullae. The second-stage juveniles are characterized by a 23.2 (22.0-24.0) µm long stylet with slightly projected or anteriorly flattened knobs, three incisures in lateral field, a 70.2 (62.5-77.0) µm long tail with bluntly rounded terminus and hyaline portion ca 58.9 (50.0-62.5)% of the tail length. Males are characterized by a 25.1 (24.5-26.3) µm long stylet with rounded knobs sloping posteriorly, four incisures in lateral field, a 29.8 (27.5-31.3) µm long spicule with bifurcate tip. Phylogenetic analysis shows that the species has unique D2D3 and ITS rRNA sequences and RFLP-ITS-rRNA profiles. Heteroderafengi n. sp. is closest to H. elachista in dendrograms inferred from both DNA sequences.

Heterodera elachista the Japanese cyst nematode parasitizing corn in Northern Italy: integrative diagnosis and bionomics

The Japanese cyst nematode Heterodera elachista was detected parasitizing corn cv Rixxer in Bosco Mesola (Ferrara Province) in Northern Italy. The only previous report of this nematode was in Asia (Japan, China and Iran) attacking upland rice; being this work the first report of this cyst nematode in Europe, and confirmed corn as a new host plant for this species. Integrative morphological and molecular data for this species were obtained using D2-D3 expansion regions of 28S rDNA, ITS1-rDNA, the partial 18S rDNA, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI), and the heat-shock protein 90 (hsp90). Heterodera elachista identified in Northern Italy was morphologically and molecularly clearly separated from other cyst nematodes attacking corn (viz. H. avenae, H. filipjevi, H. delvii, H. oryzae, H. sacchari, H. sorghi, H. zeae, Punctodera chalcoensis, and Vittadera zeaphila) and rice (H. oryzae, H. sacchari). The phylogenetic relationships of H. elachista from Northern Italy with other cyst-nematodes using rDNA and mtDNA showed a separation of the genus Heterodera in various morphospecies groups based on vulval cone structures. The development and parasitic habit of H. elachista on naturally infected corn cv Rixxer confirmed a typical susceptible reaction, including multinucleate syncytial cells in parenchymatic cells. Under greenhouse conditions, H. elachista successfully reproduced on two crops widely used in Northern Italy, such as corn (cv PR 33) and rice (cv Baldo). Considering the limited host-range of this nematode, that include two of the three world’s most important crops, special attention is needed for avoiding the dispersal of this nematode into new areas, by movement of soil on equipment, water, and contaminated containers infested soil, or agricultural practices.

Heterodera hainanensis n. sp. (Nematoda: Heteroderinae) from bamboo in Hainan Province, China – a new cyst nematode in the Afenestrata group

Heterodera hainanensis n. sp. is described and illustrated from bamboo (Phyllostachys pubescens) based on morphology and molecular analyses of rRNA D2D3 region of LSU and ITS. This new species belongs to the Afenestrata group and is characterised by lemon-shaped cyst with prominent vulval cone and vulval cone without fenestration and bullae but with weak and furcate underbridge. The second-stage juvenile (J2) has two lip annuli, a 15.8-17.5 μm long stylet with rounded knobs sloping slightly posteriorly, three incisures in the lateral field and a conical tail with hyaline portion forming 52.3-71.1% of the tail length. Heterodera hainanensis n. sp. most closely resembles species in the Afenestrata group but is distinguished from these species by a number of characters, including cyst shape and size, the presence of an underbridge, vulval slit length, J2 stylet length, stylet knobs shape, incisure number and lip annule number. Molecular analysis reveals that this species has unique LSU D2D3 and ITS rRNA sequences and RFLP-ITS-rRNA profiles, and it is most closest to H. koreana in dendrograms inferred from both markers. A key to all species of the Afenestrata group is presented.

First Report of the Cereal Cyst Nematode Heterodera filipjevi on Wheat in Serbia

The most globally recognized and economically important nematode on wheat is the cereal cyst nematode (CCN) complex (1). One of the most important species of this group is Heterodera filipjevi (Madžidov, 1981) Mulvey and Golden, 1983. During regular soil quarantine control in September 2010, Heterodera sp. cysts were found in soil samples originating from a wheat field in Gunaroš, Vojvodina Province, in northern Serbia. The wheat was a winter crop grown in a dryland production system and had an average cyst density of 2.50/100 cm3 of soil. Morphologically, the cysts were golden brown and lemon shaped with a posterior protuberance. The vulval cone was bifenestrate with horseshoe-shaped semifenestra, bullae, and underbridge. Cyst measurements (n = 30) ranged as follows: cyst length (without neck): 511.50 to 899.00 μm, cyst width: 201.50 to 682.00 μm, fenestral length: 44.80 to 65.60 μm, fenestral width: 24.00 to 40.00 μm, vulval bridge length: 12.80 to 20.80 μm, vulval bridge width: 6.40 to 14.40 μm, vulval slit: 6.00 to 12.80 μm, and underbridge length: 60.00 to 112.00 μm. The second-stage juveniles had an offset head, stylet with characteristic anchor-shaped basal knobs, four incisures, and a conical tail with a rounded tip. The J2 morphometrics (n = 30) were: length: 447.30 to 611.10 μm, width: 22.40 to 25.60 μm, stylet: 20.80 to 24.00 μm, tail length: 56.00 to 68.80 μm, tail width: 14.40 to 19.20 μm, and hyaline length: 35.20 to 44.80 μm. The ITS region was used for molecular analysis. Each DNA sample was extracted from a single cyst. Sequencing was done with primers TW81 and AB28 (2). In comparison with other H. filipjevi populations, the obtained sequence (GenBank Accession No. JX235959) revealed 99 to 100% similarity. Morphological and molecular data confirmed the existence of H. filipjevi. This is, to our knowledge, the first report of H. filipjevi from Serbia. Since wheat has important socioeconomic value for Serbia, after extensive surveys, additional phytosanitary measures may be necessary to prevent the spread of this parasite.

First Report of the Cactus Cyst Nematode, Cactodera cacti, on Cactus in Northern China.

During a survey for cyst nematodes from August to September 2011, nearly 15% of cactus (Cereus jamacaru) plants exhibited yellowing and wilting symptoms in greenhouses from Shenyang Botanical Garden, Liaoning Province, China. Cysts (averaging 50 per 100 g of samples) were detected by the sieving-decanting method on the roots and in rhizospheric soil. Second stage juveniles and eggs were isolated directly from cysts. Cysts, juveniles, and eggs were identified by morphology. Cysts (n = 12) were rounded to lemon-shaped with a protruding neck and vulva. The cyst wall had a zig-zag pattern. The vulval cone was circumfenestrate without underbridge and bullae but generally with vulval denticles. The cysts were characterized by body length excluding neck (range = 399.5 to 622.0 μm, mean = 524.9), body width (300.4 to 469.9 μm, 383.4), length to width ratio (1.1 to 1.7, 1.4), neck length (41.0 to 130.9 μm, 61.5) and width (55.0 to 98.7 μm, 76), and circumfenestral diameter length (24.6 to 30.2 μm, 28.4). Measurements of second-stage juveniles (n = 20) included length of body (range = 467.3 to 542.5 μm, mean = 513.8), stylet (23.0 to 25.8 μm, 24.6) with knobs rounded to slightly projecting anteriorly and concave on anterior surface, tail (45.9 to 59.5 μm, 52.2), and hyaline tail terminal (16.3 to 23.2 μm, 19.1). Eggs (n = 20) had heavy punctations on the shell surface. All morphological data and characteristics were consistent with Cactodera cacti (3). Molecular evidence confirmed the identification. DNA from a single cyst was extracted by using the protocol described by Subbotin et al. (2), the rDNA-internal transcribed spacer (ITS) and D2-D3 fragments of the 28S rDNA were amplified with universal primers TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3'), D2A(5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B(5'-TCGGAAGGAACCAGCTACTA-3'), respectively. The ITS PCR product was digested with eight restriction enzymes (AluI, AvaI, Bsh1236I, BsuRI, CfoI, MvaI, PstI, and RsaI) to obtain restriction fragment length polymorphism profiles (4). The ITS and D2D3 sequences were cloned and assayed using an ABI-PRISM 3730 Genetic Analyzer (Applied Sangon, Shanghai, China) and were subjected to a database search using BLAST (National Centre for Biotechnology Information). The 980-bp ITS sequence exhibited 99% similarity with that of a C. cacti isolate from Iran (GenBank Accession No. AF498393.1) and the 787-bp D2D3 sequence exhibited 99% similarity with a C. cacti isolate from Germany (GenBank Accession No. DQ328702.1). Cactus cyst nematode has been mainly reported on ornamental plants of the family Cactaceae grown in greenhouses. Infested plants become reddish brown to yellow in color, wilted, stunted, with reduced flower production and shortening flower period. With high population densities of C. cacti, death of plants may occur (1). To the best of our knowledge, this is the first report of C. cacti in northern China.

Description of a new record species of Heterodera from China (Tylenchida, Heteroderidae).

A new record species Heterodera ripae was found in the rhizosphere soil of nettle (Urtica sp.) on the bank of the Mangniu River in Shenyang City, Liaoning Province, China. The new record species can be characterized by vulval cone bifenestrate with circular or subcircular semifenestrae, vulval slit length averaging 34.2 μm, vulval bridge mostly broad, underbridge weak, bullae absent; the second-stage larvae stylet knobs rather wide and slightly projecting anteriorly, tail length averaging 43.7 μm and hyaline region length averaging 22.3 μm.

First Confirmed Report of Sugar Beet Cyst Nematode, Heterodera schachtii, in North Dakota.

Sugar beet (Beta vulgaris L.) and canola (Brassica napus L.) are major crops in North Dakota, with sugar beet production primarily in the eastern part of the state in the Red River Valley and canola production across the northern half of the state. Both crops are hosts of sugar beet cyst nematode (SBCN), Heterodera schachtii Schmidt. In April 2011, soil samples were collected from four sugar beet fields belonging to three growers who believed the fields were infested with SBCN. The fields were located in a 65-km2 area in the Yellowstone Valley of western North Dakota. Cysts were extracted by sieving and Heterodera-like cysts with eggs were observed in all four soil samples. Population densities in the four fields ranged from 100 to 1,750 eggs/100 cm3 soil. Sugar beet seedlings (cv. M832224) were grown in a potting mix for 6 weeks in the greenhouse and then transferred to conetainers (type D40; volume 656 ml) containing autoclaved river sand. Conetainers were placed in sand in plastic pots immersed in a water bath at 27°C. Three plants were each infested with 800 eggs from field No. 2. After 55 days of incubation, the average number of females was 115 per plant. A similar experiment was conducted with canola cvs. Hyclass 940, Caliber 30, and Westar, which were inoculated with 500 eggs each from field No. 2. After 53 days of incubation, there was an average of 39, 20, and 30 females for each respective cultivar. Flask-shaped cysts (n = 26) from canola roots were light to dark brown; the vulval cone was ambifinestrate with dark brown, molar-shaped bullae positioned underneath the vulval bridge. Body length (excluding neck) ranged from 600 to 850 μm (mean 701.2 μm); body width, 350 to 580 μm (mean 469.2 μm); and length/width ratio, 1.2 to 1.8 (mean 1.5). Second-stage juvenile (J2) (n = 21) body length ranged from 400 to 485 μm (mean 437.1 μm); stylet length was 25 μm (no variation) with forwardly directed knobs; conical tail with rounded tip ranged from 37.5 to 55.0 μm long (mean 46.6 μm) with hyaline region from 20.0 to 32.5 μm (mean 27.3 μm); and lateral field presented four incisures. These morphometrics were used to identify H. schachtii according to Subbotin et al. (4). Confirmation of identification was by amplification and sequencing of a 28S rDNA gene fragment (1) from individual females (GenBank Accession No. JQ040526), which was 100% identical to H. schachtii 28S rDNA sequence (GenBank Accession No. GU475088). To our knowledge, this is the first confirmed report of H. schachtii in North Dakota. A 1958 report of SBCN in North Dakota (2) was not subsequently confirmed (3). Because there is extensive canola production across the northern part of the state bordering western and eastern sugar beet- production areas, canola may serve as a bridge for movement of SBCN from west to east. SBCN is a potential threat to these two important crops.

First Report of the Cereal Cyst Nematode Heterodera latipons on Wheat in Morocco

From May to June 2011, during a survey of the wheat-growing areas in Meknes in the Saïs Region of Morocco, several cyst nematode populations were detected. Sampling was performed 1 month before wheat (Triticum durum) harvest, in fields showing patches of stunted plants. Plants were growing poorly, had chlorotic lower leaves, and a reduced numbers of ears. Root systems were short and had a bushy appearance because of increased secondary root production. No cysts were visible on the roots, but were found in the soil. Cysts were collected from soil on 200-μm sieves by the modified Cobb decanting and sieving method (1) and identified by morphology and internal transcribed spacer (ITS)-rDNA sequencing. All isolates were identified as Heterodera avenae except the isolate from Aïn Jemâa. From the latter, key morphological features from cysts and second-stage juveniles (J2) were determined. The cysts (n = 10) had the following characteristics: bifenestrate vulval cone, body length without neck 590 μm (551 to 632 μm), body width 393 μm (310 to 490 μm), neck length 75 μm (65 to 90 μm), fenestra length 64 μm (60 to 72 μm) and width 21 μm (18 to 25 μm), underbridge length 96 μm (85 to 115 μm), vulval slit length 8 μm (7 to 9 μm), vulva bridge width 27 μm (24 to 33 μm), and bullae absent. The J2s (n = 10) had the following characteristics: body length 445 μm (412 to 472 μm), body width 19 μm (19 to 21 μm), stylet length 24 μm (23 to 25 μm), four lateral lines, tail length 50 μm (46 to 54 μm), and hyaline terminal tail 28 μm (24 to 31 μm). Values of the morphological characters were within the range of H. latipons reported by Handoo (3). The bifenestrate cysts with a strong underbridge and no bullae and J2 with a tail length greater than 40 μm, a stylet longer than 15 μm, and four incisures in the lateral field were typical for H. latipons. To confirm the identification, molecular observations were made. DNA was extracted from three juveniles from three different cysts separately (4). The ITS-rDNA region was amplified using the primers 5'-CGT AAC AAG GTA GCT GTA G-3' and 5'-TCC TCC GCT AAA TGA TAT G-3' as described by Ferris et al. (2). This resulted in a 1,040-bp DNA fragment. The PCR-products were purified and sequenced (Macrogen, Inc., Seoul, Korea). All sequences obtained (GenBank Accession Nos. per cyst: JQ319035, JQ319036, and JQ319037) were compared with sequences available from the GenBank database ( www.ncbi.nlm.nih.gov ), including several species of Heterodera. This comparison revealed a sequence similarity of 97 to 99% with H. latipons and 89% or lower with any other species of Heterodera. Morphological and molecular identification demonstrated that the population of cyst nematodes from a wheat field in Aïn Jemâa, Morocco was H. latipons. In the patches with poor growing plants, 65 cysts per 100 cm3 soil were found. To our knowledge, this detection represents a new record of H. latipons. Since the nematode can cause considerable damage to wheat, one of the main cereals produced in Morocco, care should be taken to prevent the spread to other regions.

First Report of the Cyst Nematode (Heterodera elachista) on Rice in Hunan Province, China.

During a survey for the cyst nematodes (Heterodera elachista) from May to June of 2011, cyst nematodes were detected in hilly rice fields in five counties (Changsha, Pingjiang, Hengdong, Shaoyang, and Xiangxiang) of Hunan Province, China. Cyst nematodes obtained from soil samples and harvested rice root samples at these five locations had uniform morphological and molecular characteristics. Cysts (n = 20) had the following characteristics: spherical to lemon shaped, vulval cone ambifenestrate, vulval bridge narrow, medium sized underbridge, with a few dark brown bullae, body length (not including the neck) ranging from 354 to 586 μm (mean = 438.9 μm, st. dev. = 63.7); body width ranged from 283 to 495 μm (354.5, 60.1); fenestrate length of 30 to 50 μm (37.4, 5.0) and width of 25 to 47.5 μm (35.1, 7.1); underbridge length from 70 to 95 μm (83.4, 8.2); and vulval slit length from 30.3 to 55.5 μm (40.3, 9.1). J2 (n = 20) had the following characteristics: body length ranging from 404 to 525 μm (mean of 461.6 μm, st. dev. = 34.5); stylet length from 20 to 25 μm (22.5, 1.1) with rounded knob; tail length of 60 to 87.5 μm (67.3, 6.9); and hyaline terminal tail ranged from 30 to 50 μm (37.5, 6.4); lateral field with three lines. The mean and range of J2 were longer than those reported for H. elachista by Nobbs et al. (1) and Tanha et al. (4), but other morphological character values were within the range of those reported (4). DNA from a single cyst was extracted, the rDNA-internal transcribed spacer (ITS) and D2/D3 fragments of the 28S RNA were amplified with universal primers TW81 and AB28, D2A and D3B, respectively. Five ITS sequences (JN202913, JN202914, JN202915, JN202916, and JN202917) and five D2/D3 sequences (JN202918, JN202919, JN202920, JN202921, and JN202922) from nematode samples collected in Changsha, Hengdong, Shaoyang, Pingjiang, and Xiangxiang, respectively, were submitted to GenBank. These ITS sequences were remarkably similar to each other and exhibited 98.6 to 99.3% similarity with that of H. elachista isolate from Iran (AF498391), and 98.8 to 99.4% similarity with that of H. elachista isolates from Ningxia Province, China (HM560778 and HM560779). The D2/D3 sequences exhibited 99.7 to 100% similarity with that of H. elachista isolates from Ningxia Province, China (HM560842 and HM560843). These characteristics indicated that the five populations were H. elachista belonging to the 'cyperi' group (1,2). In glasshouse evaluations of the pathogenicity of these isolates, 500 second-stage juveniles were inoculated onto five 20-day-old seedlings of rice (Weiyou No.227) in 4.5-cm diameter 30-cm high tubes with six replicates. After 8 weeks, stunting and reduction of leaf length were observed and cysts were extracted from dried soil of each tube using sieves. Brown cysts (92 to 204) and white females (14 to 40) were obtained from inoculated rice from each tube. H. elachista can decrease yield by 7 to 19% and has the most severe impact during the later stages of plant growth (3). H. elachista has been previously identified from rice fields in Japan and Iran (3). To our knowledge, this is the first report of H. elachista on rice in Hunan Province, China.

Hexamermis eurygasteri n. sp. (Nematoda: Mermithidae) parasitising the sunn pest Eurygaster integriceps Puton (Hemiptera: Scutelleridae) in Turkey

A new species of mermithid nematode, Hexamermis eurygasteri n. sp. (Nematoda: Mermithidae) is described as a parasite of the sunn pest Eurygaster integriceps Puton (Hemiptera: Scutelleridae) in Turkey. The combination of the following characters separate H. eurygasteri from other members of Hexamermis Steiner, 1924, as defined by Artyukhovsky (1990) and Kaiser (1991): amphidial pouch integrated into lateral cephalic papillae; amphidial openings minute; well-developed cuticular vulval cone; small vulval lips; vagina straight or slightly curved at tip, without reverse bend; spicules shorter in length than body diameter at cloaca; spicules straight, except occasionally for short bend at base; spicule tips bluntly rounded; and two double rows of genital papillae. This is the first description of a nematode parasite of a member of the Scutelleridae and the first description of a mermithid nematode from Turkey. This mermithid has potential as a biological control agent in an integrated control programme of the sunn pest.

First Record of the Cereal Cyst Nematode Heterodera filipjevi in China.

Cereal cyst nematode (CCN) is now recognized as a widespread and often damaging parasite of wheat in China. Only Heterodera avenae has been reported in China (4). However, molecular analysis of four samples from Beijing and one from Shanxi Province indicated genetic differences from H. avenae and other named species (3). Here we report the detection of H. filipjevi at a site in Henan Province that was not included in any previous study or report. The infested crop was rainfed winter wheat (Triticum aestivum) cv. Wenmai 19 in a field near Banpopu Village in Xuchang County (34.0447°N, 113.7415°E) with a long-established maize-wheat semiannual crop rotation. During the winter growing season, the crop was patchy with uneven growth and cyst nematode females were observed on the roots. In June 2009, soil was collected and mature cysts were extracted for morphological and molecular identification. Cysts were also kept at 4°C for 2 months and then incubated in shallow water at 15°C for a month to obtain second-stage juveniles (J2). Measurements (range; mean ± sd) of 10 cysts were body length including neck (569 to 786 μm; 699 ± 56), body width (403 to 600 μm; 523 ± 55), length:width ratio (1.3 to 1.5; 1.3 ± 0.1), neck length (61 to 125 μm; 106 ± 19) and width (49 to 83 μm; 69 ± 13), fenestra length (52 to 59 μm; 57 ± 2.9) and width (24.5 to 34.4; 27.9 ± 3.5), underbridge (64 to 101 μm; 85 ± 10), and vulval slit (7.4 to 10.0 μm; 9.6 ± 1.0). Lemon-shaped cysts were brown with a surface zigzag pattern. The vulval cone was bifenestrate with horseshoe-shaped semifenestra, with heavy underbridge and many bullae. The J2 (n = 22) measurements were body length (496 to 590 μm; 552 ± 24), body width (20.0 to 23.8; 21.5 ± 0.9), stylet (22.8 to 25.3; 24.0 ± 1.0) with anchor-shaped basal knobs, tail (47 to 64; 61.6 ± 4.4), and hyaline tail terminus (32 to 43; 40.2 ± 3.0). The J2 had up to four lateral lines, but the inner two were often the only lines clearly visible, and the shape of the stylet knobs, tail, and tail terminus were consistent with H. filipjevi. All morphological data and characters were consistent with H. filipjevi (1). Specimens have been lodged with the Australian National Insect Collection. DNA from single cysts was extracted to amplify the internal transcribed spacer region of rDNA by PCR with forward primer TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and reverse primer AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3') (2). The PCR product was sequenced (Genbank Accession No. HM027892) and digested by restriction enzymes (AluI, CfoI, HaeI, HinfI, PstI, RsaI, TaqI, and Tru9I) to obtain restriction fragment length polymorphism profiles (2). Profiles for the Xuchang population consistently matched those published for H. filipjevi and were distinct from those of H. avenae and other species (3). Phylogenic analysis of the sequence further indicated conspecificity with H. filipjevi. These morphological and molecular data confirmed that the specimens from Xuchang were H. filipjevi, which represents the first detection of H. filipjevi in China, and extends the known distribution of the species from Europe, North America, South Asia, and West Asia to East Asia. This finding adds complexity to the management of CCN in China, especially for control by host resistance, which now must consider both species and pathotype diversity.

IDENTIFIKASI KARAKTER MORFOLOGI NEMATODA SISTA PADA TANAMAN JAGUNG (Heterodera zeae) DI INDONESIA

Identification of nematode species is the first step in the resolution of major nematological problems. Accurate identification of maize cyst nematode species is essential for more detailed research, particularly for it control measure. Although morphological observations by using the key characters of each nematode are suitable for identification purposes, the cyst nematodes could not be clearly identified based on it due to the wide variations. For the identification of the cyst nematode species, key characters relating to body surface, head, mouth, esophagus, tail as well as habitat and feeding habits was used in the present examination. Procedures for the permanent and temporary preparations of nematodes were used. The nematode specimens were observed under the microscope to confirm the key characters. Results of measurements and descriptions revealed that the cyst nematode collected from maize roots in Madura island have the same ambifenestrate as that of Heterodera zeae. The underbridge is short and thin and there is two layer of bullae arrangement in vulval cone, the first layer with four finger-like bullae located immediately below the underbridge and the second layer very randomly located below the first one. These characteristics are of H. zeae. These complete of morphological identifications are a new report of H. zeae in Indonesia.

Heterodera glycines

<i>Heterodera glycines</i>