Heterodera Species Research Articles

Analyses of Fungal Communities from Culture-Dependent and -Independent Studies Reveal Novel Mycobiomes Associated with Globodera and Heterodera Species

Plant-parasitic nematodes are important pathogens of agricultural crops. Of particular importance are cyst nematode species of Globodera and Heterodera, which remain a major constraint to global production of crops such as potato, soybean, and wheat. Recent restrictions on the use of some synthetic nematicides has created an urgent need for alternative methods of controlling cyst nematodes. This project aimed to (i) document fungi found associated with cyst nematodes, (ii) determine if the fungi associated with cyst nematodes are dependent on growth conditions or nematode species, (iii) identify core mycobiomes for each of the eight cyst nematode species, (iv) identify the core mycobiome across all cyst nematode species studied, and (v) determine if the fungi associated with Globodera species are structured geographically. Sixty cyst nematode populations from eight species— Globodera sp., G. ellingtonae, G. pallida, G. rostochiensis, H. avenae, H. carotae, H. glycines, and H. schachtii—were collected from several geographic locations globally and screened for fungi using both culture-dependent and -independent methods. The core mycobiome was determined for each species. In total, 744 species of fungi were found to be associated with cyst nematodes. Fungal species shared across all cyst nematode species included Pochonia sp., Sporothrix sp., Neocosmospora solani, Exophiala equina, Mortierella sp., and Sarocladium kiliense. The core mycobiomes were conserved between cyst nematode species irrespective of their geographic origin. Cyst nematodes collected from the field or from greenhouse cultures also recruited similar core mycobiomes. These findings could be relevant when choosing fungi to evaluate as biocontrol agents against cyst nematodes.

Influence of Seasonal Variations on Population Dynamics of Phyto-Parasitic Nematodes in Soil and Roots of Bell Pepper (Capsicum annuum L.) in Otari, Rivers State, Nigeria

Weather instability affects nematode proliferation in soil, affecting plant growth. Identifying endemic species and favourable seasons can guide farmers in cultivating crops during specific seasons, minimizing infections and improving yield. A survey to evaluate the influence of seasonal disparity on plant parasitic nematodes population in soil and roots of bell pepper (Capsicum annuum L.) was carried out during dry and rainy seasons in Otari, Abua/Odual Local Government Area of Rivers State, Nigeria. Five bell pepper cultivated fields were randomly selected and a total of 300 soil samples were collected and assayed for soil nematodes. The soil samples were collected using a soil auger at 0-15 cm depth, and roots were collected by the use of sterilized kitchen knife simultaneously for the isolation of nematode. The modified sieve plate method was employed for the extraction of nematodes. Nematode identification was carried out using a pictorial key. A total of 2,220 nematodes from 11 genera were reported in this study, among which 1,290 (58.1%) were recovered during the rainy season while the dry season showed 930 (41.9%) nematodes. Nematode population vary significantly between seasons in soil (P = .13) and roots of bell pepper (P = 3.90), with Heterodera species having higher density (1.44 and 1.03) during the rainy and dry seasons respectively in the soil. High density of Meloidogyne (0.97) was recorded during the rainy season and Radopholus spp (1.40) during the dry season in the root of bell pepper plant. The study showed that seasonal variations impacted on nematode population abundance in fields, establishing that nematodes are responsive to every unsteady condition of the soil environment resulting from seasonal disparity, and only species who adapt rapidly survive. Farmers should increase crop cultivation during the rainy season to boost yield, as nematodes population decline as rainfall increases.

DNA barcoding of Australian cereal cyst nematode populations with comments on likely origin and taxonomy (Tylenchoidea: Heterodera)

A species of Heterodera has been known to parasitise cereals in Australia since the 1930s. It caused significant yield losses across Australia’s cereal growing regions until resistance breeding largely brought it under control, although it still occurs occasionally, especially in South and Western Australia. Australian cereal cyst nematode has long been considered to represent Heterodera avenae. However, in 2002 the name Heterodera australis was proposed for Australian cereal cyst nematode, as it could be distinguished from all non-Australian populations of H. avenae via biochemical and molecular methods. This new species proposal came with speculation that both H. avenae and H. australis might occur in Australia, and that H. australis might represent a native species. The name H. australis has generally not been accepted by Australian scientists, nor the notion that it is native. There remains some uncertainty as to the validity of H. australis and whether more than one species of cereal cyst nematode occur in Australia. Using a molecular barcoding approach (COI, 18S, ITS, 28S) we examined the species composition of cyst nematodes present in soil samples collected between 1989–2023 from Australian cereal growing regions. We find only one species of Heterodera parasitising cereals and, based on phylogenetic analyses, accept the validity of H. australis as the name best representative of this species. We also argue that, based on presently available evidence, H. australis is not native and was most likely introduced into Australia from Asia in the 1850s, rather than from Europe as has been generally assumed.

The occurrence of cyst nematodes in agricultural fields of Southern Manitoba

In southern Manitoba, Canada, a survey was carried out in 2012 and 2013 to determine the presence of Heteroderidae cyst-forming plant-parasitic nematodes, with a focus on the soybean cyst nematode (SCN) (Heterodera glycines). A total of 48 fields having grown soybean were sampled. A modified Fenwick elutriation-flotation technique was used to extract cysts with a 75% cyst recovery efficiency. Cyst population density averaged 0.9 cysts kg−1 soil, with a total of 65 cysts recovered. Preliminary screening of cysts, based on general body shape and vulval cone top structure, showed the presence of cysts belong to circumfenestrate, and ambifenestrate groups of cyst-forming nematodes. Limited morphological data was accessible due to poor quality or insufficient cysts for analysis; however, generated DNA sequences for nuclear rDNA ITS and D2-D3 expansion region of the 28S rRNA were obtained for four samples and matched sequences in GenBank for the cyst nematodes Cactodera milleri, C. torreyanae, C. weissi, C. estonica and unknown Cactodera species. Only one of the ambifenestrate cysts with a cone top structure of Heterodera species yielded DNA for analysis and its identification was ambiguous for soybean cyst nematode (SCN). None of the cysts were positive through SCN diagnostic PCR. Cactodera is not a pest of soybean or other crops in Manitoba. These cyst nematodes are likely to be naturally associated with weeds and grasses in the sampled fields or may be introduced from neighbouring states of the USA. Further annual surveys are needed and recommended in the near future to encompass more soybean fields and corroborate the absence of the pest.

Morphological and molecular identification of cyst nematode species (Heterodera spp.) in Algerian cereal fields

Cereal cyst nematodes (Heterodera spp.) are distributed globally and cause severe production losses of small grain cereals. To investigate the occurrence of cereal cyst nematodes in wheat-growing areas of Algeria, a survey was conducted and 27 cereal cyst nematode populations were collected. The populations were initially identified based on their morphological and morphometric characters, followed by molecular methods using speciesspecific primers, complemented by ITS-rDNA sequences. The morphological and morphometric features of second-stage juveniles (J2s) and cysts supported the presence of three Heterodera species: H. avenae, H. filipjevi and H. hordecalis. All morphological values of these distinct populations were very similar to those previously described for these species. Using species-specific primers for H. avenae and H. filipjevi, the specific bands of 109 bp and 646 bp confirmed the morphological identification of both species, respectively. In addition, the internal transcribed spacer (ITS) regions were sequenced to study the diversity of the 27 populations. These sequences were compared with those of Heterodera species available in the GenBank database (www.ncbi.nlm.nih.gov) and re-confirmed the identity of the species. Nineteen sequences of ITS-rDNA were similar (99–100%) to the sequences of H. avenae published in the GenBank, six sequences were similar (99–100%) to H. hordecalis, and two were similar (98–99%) to H. filipjevi. The results of this study are of great value to breeding programs and extension services, where they will contribute to the design of control measures to keep damaging nematodes in check.

Reliability and Utility of Standard Gene Sequence Barcodes for the Identification and Differentiation of Cyst Nematodes of the Genus Heterodera

Difficulties inherent in the morphological identification of cyst nematodes of the genus Heterodera Schmidt, 1871, an important lineage of plant parasites, has led to broad adoption of molecular methods for diagnosing and differentiating species. The pool of publicly available sequence data has grown significantly over the past few decades, and over half of all known species of Heterodera have been characterized using one or more molecular markers commonly employed in DNA barcoding (18S, internal transcribed spacer [ITS], 28S, coxI). But how reliable are these data and how useful are these four markers for differentiating species? We downloaded all 18S, ITS, 28S, and coxI gene sequences available on the National Center for Biotechnology Information (NCBI) database, GenBank, for all species of Heterodera for which data were available. Using a combination of sequence comparison and tree-based phylogenetic methods, we evaluated this dataset for erroneous or otherwise problematic sequences and examined the utility of each molecular marker for the delineation of species. Although we find the rate of obviously erroneous sequences to be low, all four molecular markers failed to differentiate between at least one species pair. Our results suggest that while a combination of multiple markers is best for species identification, the coxI marker shows the most utility for species differentiation and should be favored over 18S, ITS, and 28S, where resources are limited. Presently, less than half the valid species of Heterodera have a sequence of coxI available, and only a third have more than one sequence of this marker.

Heterodera amaranthusiae n. sp. (Nematoda: Heteroderidae), a new cyst nematode parasitising Amaranthus retroflexus L. in China

Summary A new species of cyst-forming nematode, Heterodera amaranthusiae n. sp., is described and illustrated from the weed, Amaranthus retroflexus, in a potato field in Yunnan Province, China. It is characterised by having canary to russet-brown and asymmetric lemon-shaped cyst, distinct neck, bifenestrate vulval cone, relatively short vulval slit of 29 (28-32) μm, bullae absent and underbridge absent or weak if present. Second-stage juveniles are characterised by a well-developed stylet of 23 (22-25) μm with robust shaft and basal knobs concave anteriorly, tail conoid, 51 (48-58) μm long and hyaline region comprising 48 (41-53)% of its length. Morphologically and morphometrically it most resembles H. vallicola in the Humuli group. The ITS, 28S and COI gene sequences of H. amaranthusiae n. sp. clearly differentiate it from other Heterodera species. For diagnostic purposes, restriction enzyme analysis of the ITS region and three restriction enzymes, AluI, BsuRI (HaeIII) and CfoI (HhaI), were selected, clearly distinguishing H. amaranthusiae n. sp. from representative species in the Humuli group. Phylogenetic relationships with other species of the genus, inferred from two ribosomal regions and the cytochrome oxidase c subunit 1 region, based on Bayesian analysis, consistently showed that H. amaranthusiae n. sp. clustered with high support with other Humuli group species but with separate species status.

Differential Morphological, Structural and Biological Characteristics of Cysts in Heterodera Species in Korea.

Morphological (cyst shape, color, and sizes [length (L), maximum width (W), volume and “a” (L/W)]), structural (vulvar cone slope angle [VCSA], surface wrinkle [VCSW], cyst wall thickness, composition, and texture) and biological characteristics (fecundity, hatching, and emergence [number of second-stage juveniles (J2) from a cyst]) in preceding Heterodera glycines (Hg), currently-recorded H. sojae (Hs) and H. trifolii (Ht) were examined by microscopy. Cysts were lemon-shaped, indicating the genus is Heterodera except for Hs that formed frequently globular cysts with significantly flatter VCSA (102.2°) with smooth VCSW than Hg (50.6°) and Ht (82.0°), but not genus Globodera because of the presence of vulvar cone in Hs. Ht was significantly larger in all morphological characteristics than Hg and Hs, suggesting Ht may be diagnosed differentially by cyst sizes and also host plant preferences. Hs showed smaller “a” value with more globular shape and stronger structures with more thickened and strengthened collagen-like texture of cyst wall than Hg and Ht. This suggests Hs may be diagnosed differently by structural characteristics from the others, especially Hg with similar cyst sizes. There were no significant differences in emergence (inoculum potential) among cyst nematodes due to the offset of fecundity and hatching rate; however, the inoculum potential of Hs may be not so persistent as Hg and Ht in fields because of its lower fecundity and higher hatching rate (causing rapid inoculum loss) than the others. These characteristics of cysts provide information useful for simple and differential diagnoses and reliable management of cyst nematodes.

Interacciones fisiológicas de plantas con nematodos fitoparásitos: una revisión

Los nematodos fitoparásitos se encuentran entre los patógenos de plantas más perjudiciales a través del mundo, afectando el crecimiento y rendimiento de los cultivos. La respuesta de las plantas al daño ocasionado por los nematodos está estrechamente relacionada con su ubicación donde se alimentan, como ectoparásitos o endoparásitos, y los tipos de daño celular que producen, tales como la destrucción de células ocasionada por especies de Pratylenchus y Radopholus; la sincitia, por especies de Heterodera y Globodera; y la formación de células gigantes, por especies de Meloidogyne. El objetivo de esta revisión es analizar el alcance de los resultados de investigaciones sobre el entendimiento de las implicaciones fisiológicas que tiene la interacción de la planta con nematodos fitoparásitos, principalmente los que destruyen la célula, forman sincitia y células gigantes. En general, los nematodos reducen la absorción de agua a través de las raíces de las plantas y crean un desbalance de macro y micronutrimentos que inducen cambios fisiológicos en estas y en los procesos fotosintéticos. Los nematodos fitoparásitos incluidos en esta revisión, independiente del tipo de daño que ocasionan, reducen las funciones fisiológicas de las raíces y de la parte aérea produciendo pérdidas considerables en rendimiento. Se requiere más investigación que relacione el estado nutricional de las plantas con las prácticas de nutrición edáfica y foliar, que conlleve a una mejor respuesta fisiológica del hospedante, lo cual permitiría un menor impacto del parasitismo de los nematodos en las plantas, contribuyendo al manejo integrado de estos

Description of Heterodera microulae sp. n. (Nematoda: Heteroderinae) from China - a new cyst nematode in the Goettingiana group.

A new cyst-forming nematode, Heterodera microulae sp. n., was isolated from the roots and rhizosphere soil of Microula sikkimensis in China. Morphologically, the new species is characterized by lemon-shaped body with an extruded neck and obtuse vulval cone. The vulval cone of the new species appeared to be ambifenestrate without bullae and a weak underbridge. The second-stage juveniles have a longer body length with four lateral lines, strong stylets with rounded and flat stylet knobs, tail with a comparatively longer hyaline area, and a sharp terminus. The phylogenetic analyses based on ITS-rDNA, D2-D3 of 28S rDNA, and COI sequences revealed that the new species formed a separate clade from other Heterodera species in Goettingiana group, which further support the unique status of H. microulae sp. n. Therefore, it is described herein as a new species of genus Heterodera; additionally, the present study provided the first record of Goettingiana group in Gansu Province, China.

Identification of Heterodera glycines (Tylenchida; Heteroderidae) Using qPCR.

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea’s soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R2 = 0.996, slope = −3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

Occurrence of Neodolichodorus persiangulfus n. sp. (Nematoda: Dolichodoridae) in mangrove forests of southern Iran

AbstractAn infestation of mangrove forests with Neodolichodorus persiangulfus n. sp. in southern Iran is reported. This paper is the first to report a representative species of the genus in the country. The new species was characterized by 2,120 (1795–2400) µm long females, and 1755 (1533–2019) µm long males, a continuous to slightly offset cephalic region separated from the rest of the body by a shallow depression, having 5–7 annuli and a distinct labial plate, a lateral field with four lines, the outer ones crenate, irregularly areolated in anterior and posterior body region, 79 (66–85) μm long female stylet, a didelphic‐amphidelphic female reproductive system with asymmetric sclerotized vaginae, phasmids 5–10 annuli behind the anus, female tail elongate conoid, suddenly narrowing at 5–12 annuli posterior to anus and males with trilobed bursa, 52.5 (47–58) μm long spicules and 21.5 (18–27) μm gubernaculum. With the elongate‐conoid female tail and cʹ >1.2, the new species is close to five known species of the genus: N. brevistylus, N. leiocephalus, N. paralongicaudatus, N. rageshi and N. sinensis, but is distinguished from them by the morphology of the female tail, areolation type of the lateral field, position of hemizonid and phasmid and morphometric data. Molecular phylogenetic analyses using sequences of the near full‐length fragment of the small and the D2‐D3 expansion segments of the large subunit ribosomal DNA (SSU and LSU rDNA D2‐D3) revealed the new species placed in a clade including one isolate of Neodolichodorus sp. and some species of Globodera, Heterodera and Rotylenchulus with 0.89 Bayesian posterior probability (BPP) in SSU phylogeny. The only available SSU sequence of the genus was the tentative closest relative to the new species in the inferred SSU tree. In the reconstructed LSU phylogenetic tree, N. persiangulfus n. sp. formed a poorly supported clade with Rotylenchulus spp. in Bayesian inference (BI), and the only other available D2‐D3 sequence of the genus occupied a distant placement to the new species.

Morphological and Molecular Characterization of Heterodera schachtii and the Newly Recorded Cyst Nematode, H. trifolii Associated with Chinese Cabbage in Korea

The sugar beet cyst nematode, Heterodera schachtii is a well known pathogen on Chinese cabbage in the highland fields of Korea. However, a race of cyst forming nematode with close morphological resemblance to H. trifolii was recently isolated from the same Chinese cabbage fields. Morphological species differentiation between the two cyst nematodes is challenging, with only minor differences between them. Thus, this study described the newly intercepted H. trifolii population, and reviewed morphological and molecular characteristics conceivably essential in differentiating the two nematode species. A comparison of morphometric measurements of both infective juveniles and vulval cones of cysts showed significant differences between the two cyst nematodes. When total RNA and genomic DNA were extracted from a mixed field population, COI genes and ITS regions were clearly amplified with primers of the two Heterodera species, suggesting that Heterodera population collected from the Chinese cabbage field consisted of a mixture of two species. COI and ITS of H. trifolii were predominantly amplified from nucleotides prepared from H. trifolii monoxenic population whereas those of H. schachtii were strongly detected in H. schachtii monoxenic cultures. Thus, this study confirms the coexistence of the two species in some Chinese cabbage fields; and the presence of H. trifolii in Korea is reported here for the first time.

Morphological and molecular characterisation of Heterodera filipjevi (Madzhidov, 1981) from the Slovak Republic

Cysts, males and juveniles from a population of Heterodera filipjevi found on sports-ground turf in Trnava, Slovak Republic, in 2016 are described based on morphology and morphometrics. The identity of species of juveniles extracted from cysts was subsequently determined and confirmed by PCR-RFLP and sequencing. Morphologically, cysts, juveniles and males from the Slovak Republic are similar to paratypes from Tajikistan. The results of the phylogenetic analysis of the ITS rRNA gene sequences confirmed the species identification and phylogenetic relationship of H. filipjevi with other Heterodera species. Of interest, this analysis showed the close similarity between the Slovakian (KY349106) and Chinese (KU896216) samples, which differed by a single nucleotide and clustered together. To our knowledge, this is also the first report of H. filipjevi from the Slovak Republic, thus increasing the total number of cyst nematode species of the Heterodera genus known to occur in the country to a total of 15 species.

Morphological and molecular characterisation of Heterodera koreana (Vovlas, Lamberti & Choo, 1992) Mundo-Ocampo, Troccoli, Subbotin, Del Cid, Baldwin & Inserra, 2008 (Nematoda: Heteroderidae) from bamboo in Japan

The Korean cyst nematode, Heterodera koreana, was recorded for the first time from Japan and characterised morphologically, morphometrically and molecularly. In total, 41 populations were detected from soil samples collected from the rhizosphere of four bamboo species in Japan: 31 populations from moso bamboo (Phyllostachys edulis), seven from madake (P. bambusoides), two from henon bamboo (P. nigra var. henonis) and one from fish pole bamboo (P. aurea). The morphology and morphometrics of the Japanese population were in agreement with those of the original description of H. koreana from South Korea and other subsequent descriptions from China and Iran, with the exception of some minor differences. The results of the phylogenetic analyses of the D2-D3 expansion segments of 28S rRNA gene and ITS rRNA gene sequences confirmed the species identification and phylogenetic relationship of H. koreana with other Heterodera species. The COI mtDNA gene sequences were obtained for the first time for H. koreana. Three COI haplotypes found in Japanese H. koreana populations showed a characteristic geographical distribution in Japan.

The impact of resistant and susceptible wheat cultivars on the multiplication of Heterodera filipjevi and H. avenae in parasite‐infested soil

Cereal cyst nematode (CCN) severely threatens wheat production in many regions of China. Cultivars susceptible to CCN are the main reason for its spread. This study was initiated to determine whether wheat cultivars conferring different resistance levels are the dominant determinants of CCN populations in the rhizospheric soil. Field experiments were conducted at two locations in Henan province, China, where high populations of Heterodera filipjevi or H. avenae have occurred, during the growing seasons of 2010/11 and 2011/12. Conventional enumeration of white female nematodes on the plant roots, reproductive factor (Rf) and a molecular diagnostic approach, PreDicta B test, a soil testing service based on a sensitive quantitative PCR technique, were used to determine the nematode populations in the rhizospheric soils of seven common wheat and durum wheat cultivars with different reactions to CCN. The resistant responses to CCN conferred by durum wheat Wascana and Wakooma and common wheat Madsen were effective against both H. filipjevi and H. avenae and resulted in significantly fewer nematodes (<5 females) on the roots, and lower Rf. Those cultivars were effective in limiting nematode propagation, resulting in fewer CCN eggs in their rhizospheric soils. Taikong 6 conferred moderate resistance (5–10 females) to both Heterodera species. Tianmin 668 only showed resistance to H. avenae. Aikang 58 and Wenmai 19 were susceptible to both CCN species, which facilitated increases in the nematode populations. These results demonstrate that the reactions to CCN of wheat genotypes have obvious impact on the propagation of nematodes.

Distribution of the Cereal Cyst Nematodes (Heterodera spp.) in Wheat and Barley Fields in North-Eastern Regions of Syria

Cereal cyst nematodes (CCN) are important plant-parasitic nematodes of wheat and barley, and exist in most of the cereal growing regions of the world. As there is limited information on the nature and distribution of CCN species in Syria, a survey was conducted in north-eastern Syria to assess the distribution of CCN in the main wheat and barley growing areas. In the summer of 2009, a total of 167 composite soil samples were collected from 167 wheat and barley fields. Cysts were extracted from soil using the Fenwick can technique, then quantified and identified up to the species level by using both morphological and molecular methods. The study revealed that 62% of the fields were infested with the three Heterodera species: H. avenae, H. filipjevi and H. latipons. However, the most prevalent species was H. latipons, which was present in 76% of the infested samples, while it occurred alone in 67% of those samples. Heterodera avenae was detected singly in 20% of the samples, while 11% consisted of mixed populations with other species. Heterodera filipjevi was never detected alone; it was found mixed with H. avenae and/or H. latipons in 9% of the samples. The high number of extracted cysts (up to 116 cysts × 200 g−1 of soil) is most likely related to the monoculture practiced in that region and the fact that growers are unaware of the existence of resistant varieties.

Development of qPCR assays for quantitative detection of Heterodera avenae and H. latipons

Twelve Heterodera species are considered of major economic significance in cereals, of which Heterodera avenae, H. latipons and H. filipjevi are the most important. Precise identification and quantification of these nematodes are necessary to develop effective integrated pest control. This study reports on the use of the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop qPCR assays that could be used for the identification and quantification of H. avenae and H. latipons. Two qPCR primer sets, each comprising two primers and a probe, were designed for each of both species. After optimization, the qPCR assays using a single second-stage juvenile (J2) were able to identify and quantify H. avenae and H. latipons. Their specificity was confirmed by the lack of amplification of J2 of 14 other Heterodera species. A qPCR using DNA extracted from 120 J2 + eggs of H. avenae and H. latipons resulted in steady Ct-values (Ct = 22.33 ± 0.1 and Ct = 21.83 ± 0.12, respectively). Dilution series of DNA extracted from 120 J2 + eggs of the two species were made. The assays for both species resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R2 = 0.99; slope = −3.03 and R2 = 0.99; slope = −3.28 for H. avenae and H. latipons, respectively). The two qPCR assays provide a sensitive and valid tool for rapid detection and quantification of the two species whether they occur alone or in mixtures with other species.

Morphological and molecular identification of cereal cyst nematodes from the eastern Mediterranean region of Turkey

The morphological and molecular characteristics of 41 populations of cereal cyst nematodes (Heterodera avenae group) collected in Adana, Osmaniye, Kahramanmaras, Hatay, Gaziantep, and Kilis provinces in the eastern Mediterranean region of Turkey were studied. The morphological characters and morphometric features of second-stage juveniles and cysts showed the presence of 3 Heterodera species: H. avenae, H. filipjevi, and H. latipons. All morphological values of these distinct populations were very similar to those previously described for these species. Genetic variation was observed among the identified cyst nematode species H. avenae, H. filipjevi, and H. latipons. Intraspecific polymorphism was observed within H. avenae and H. latipons but not in H. filipjevi populations. Molecular analysis using ITS regions of rDNA confirmed the identities of the 3 Heterodera species. According to our results, 75% of isolates were identified as H. avenae, 15% as H. latipons, and 10% as H. filipjevi.

Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi

Twelve Heterodera species are of major economic significance in wheat and barley. Of these, H. avenae, H. filipjevi and H. latipons are among the most important ones, and sometimes coexist. The identification of Heterodera species using morphological characteristics is time consuming, requires specialized skill and can be imprecise, especially when they occur mixed in field populations. Molecular techniques can provide a more accurate way for nematode identification. This study reports the results of experiments targeting the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop species-specific primers that could be used for the identification of H. avenae and H. filipjevi. The COI gene of 9 Heterodera spp. and Punctodera punctata was partially sequenced and the resultant sequences were aligned to find unique sites suitable for the design of primers. The alignment showed variability between H. avenae, H. filipjevi and other Heterodera species. Two sets of species-specific primers were identified for the identification of both species and the conditions for their use in PCR were optimised. The specificity of the designed primers was checked by comparison with one population of P. punctata and populations of 14 other Heterodera species, nine populations of H. avenae and 10 populations of H. filipjevi originating from different countries. To test the sensitivity, the PCR was run with DNA extracted from five second-stage juveniles (J2) of H. avenae or five J2 of H. filipjevi mixed with DNA extracted from varying numbers of J2 of H. latipons. It was possible to detect as few as five J2 of H. avenae or H. filipjevi among 100 J2 of H. latipons. The two primers sets allow the detection of H. avenae and H. filipjevi where they occur in mixed populations with other Heterodera spp.