Heterodera filipjevi continues to be a major threat to wheat production worldwide. Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease. In the present study, a TaqManminor groove binder (TaqMan-MGB) probe-based fluorescence quantitative real-time PCR (qPCR) was successfully developed and used for quantifying H. filipjevi from DNA extracts of soil. The primers and probe designed from the obtained RAPD-SCAR marker fragments of H. filipjevi showed high specificity to H. filipjevi using DNA from isolatesconfirmed species of 23 Heterodera spp., 1 Globodera spp. and 3 Pratylenchus spp. The qPCR assay is highly sensitive and provides improved H. filipjevi detection sensitivity of as low as 4^–3 single second-stage juvenile (J2) DNAs, 10^–3 female DNAs, and 0.01 μg μL^–1 genomic DNAs. A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H. filipjevi in naturally infested field soils. There was a high correlation between the H. filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay. qPCR potentially provides a useful platform for the efficient detection and quantification of H. filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils

The sugar beet cyst nematode, Heterodera schachtii, is a major parasite of sugar beet which has been recognized and listed as a quarantine nematode in China and more than 20 countries and regions worldwide.  A survey for important nematodes was undertaken in the sugar beet planting area of China during 2015–2018, and numerous cysts were collected from sugar beet fields in Xinyuan County, Xinjiang Uygur Autonomous Region of China.  The observations of morphological and morphometric characteristics revealed that cysts, vulval cones and second-stage juveniles of the Xinjiang population were in the same range of each other and within those of other reported H. schachtii populations.  Molecular analysis of rDNA-ITS, 28S-D2/D3 and mtDNA cytochrome c oxidase subunit 1 (COI) gene sequences suggested that the Xinjiang population clustered in a branch with those foreign populations, and the sequence similarity was as high as 99.81–100%.  Moreover, this result was confirmed by PCR assay with species-specific primer SHF6 and rDNA2 of H. schachtii, and the pathogenicity test confirmed successful Xinjiang population reproduction in both plant hosts.  In conclusion, based on morphological and molecular characterization, this study confirmed that the cyst nematode population collected from sugar beet fields in Xinjiang is H. schachtii.  As far as we know, this is the first report of H. schachtii on sugar beets in Xinjiang, China.

The potato cyst nematodes (PCN) Globodera rostochiensis (Wollenweber) Skarbilovich, 1959 is considered the most damaging nematode pest of potato worldwide that causes significant yield losses, and this nematode is recognized and listed as a quarantine nematode in many countries (EPPO 2017).  China is currently the largest producer of potato in the world, while the total production is also the highest (Guan and Cai 2019).  The survey for cyst nematodes on potato were conducted in Yunnan and Sichuan provinces of China during 2018–2020, numerous cysts were observed on potato roots in Huize County and Ludian County of Yunnan Province, Zhaojue County and Yuexi County of Sichuan Province.  Cysts and second-stage juveniles (J2s) were isolated from each soil sample using the Cobb decanting and sieving method.  The morphology of cysts and J2s and molecular analysis established the identity of this species as golden cyst nematode Globodera rostochiensis (Subbotin et al. 2010).  For morphological analysis, the cysts were characterized by smoothly rounded with a small projecting neck, brown and golden color, terminal cone was absent and circumfenestrate.  The key morphometrics of cysts (n=25) were: length excluding neck 705±24 (689–747) μm, width 698±28 (678–759) μm, number of cuticular ridges between anus and vulval fenestra 17.3±1.7 (14–19); fenestral diameter 13.6±1.1 (12.25–15.45) μm; distance from anus to the edge of fenestra 63.7±11.3 (48.23–79.14) μm; Granek’s ratio 4.7±0.7 (3.92–5.75).  The key morphometrics of J2s (n=25): body length 453.9±16.6 (440–496) μm, stylet length 21.9±1.0 (20.3–24.3) μm, tail length 51.1±3.2 (45.5–55.5) μm, and hyaline region length 24.4±2.5 (21.7–29.9) μm.  Morphology of the cysts and J2 were consistent with those of G. rostochiensis (Subbotin et al. 2010; EPPO 2017).  Moreover, the identification result was confirmed by PCR using universal primers TW81 (5´-GTTTCCGTAGGTGAACCTGC-3´) and AB28 (5´-ATATGCTTAAGTTCAGCGGGT-3´) for ITS region and D2A (5´-TTTTTTGGGCATCCTGAGGTTTAT-3´) D3B (5´-AGCACCTAAACTTAAAACATAATGAAAATG-3´) for rDNA-28S region, respectively.  The ITS rDNA sequences (GenBank accessions MZ042365, MZ042366, MZ042369, and MZ042370) exhibited 99.83% identity match to G. rostochiensis sequences available in the GenBank (GQ294513).  Sequence from the 28S region (GenBank accessions MZ057595, MZ057596, MZ057599, and MZ057600) was 99.33% similar to those of G. rostochiensis isolate from MF773722.  The species was also confirmed with species-specific primers ITS5 (5´-GGAAGTAAAAGTCGTAACAAGG-3´) and PITSr3 (5´-AGCGCAGACATGCCGCAA-3´) (Bulman and Marshall 1997), a single 434-bp fragment was obtained from Huize, Ludian, Zhaojue and Yuexi populations.  The pathog enicity testing of Huize, Ludian, Zhaojue and Yuexi, three weeks-old potato plants (cv. Qinshu 9)

were inoculated with 2 000 eggs, and cultured in an incubator at 23°C/20°C with a 16 h/8 h light/dark photoperiod.  After three months inoculation, 36±7.2 cysts and females were extracted from the infested potato roots, no females and cysts were observed on control plants.  

This is the first report of potato golden cyst nematode G. rostochiensis in China.  

Stunt nematodes (Tylenchorhynchus spp.) are obligate migratory root ecto-parasitic nematodes found in the fields of many cultivated crops.  These nematodes, with phyto-sanitary potential, are frequently ignored or misdiagnosed as pests, and this may pose a threat to food security.  The accuracy of its identification based on a morphological approach has been challenged recently, due to the overlapping of the morphological and morphometric characters of the species.  Consequently, the objective of this study is to identify and characterize stunt nematodes present in 54 fields cultivated with cereal crops (wheat, maize and rice) in the savannahs of northern Nigeria, using integrative taxonomy and molecular approaches.  The molecular and morphological studies identified and confirmed the presence of T. annulatus as the occurring specie in the savannahs of northern Nigeria.  The phylogenetic analysis was carried out using the internal transcribed spacer (ITS) and 28S genes of ribosomal DNA further confirmed the presence of T. annulatus.  The first molecular characterization and sequences of the ITS and 28S rDNA gene for T. annulatus from Nigeria were provided by this research.  Also, according to our literature search, this is the first report on T. annulatus in wheat, maize and rice in the savannahs of northern Nigeria.  Further study to test the pathogenicity of the parasitic nematode species found in this survey is recommended for the prioritization and development of efficient management strategies.

Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province, China.  In order to establish a rapid, reliable and specific molecular detection method for M. vitis, the species-specific primers were designed with rDNA-ITS (ribosomal DNA internal transcribed spacer) gene fragment as the target.  The reaction system was optimized and the reliability, specificity and sensitivity of primer were testified, therefore, a rapid PCR detection method for M. vitis was established.  The result showed that the optimal annealing temperature of the primers was 53°C, which was suitable for the detection of different life stages of M. vitis.  Specificity test showed that the specific fragment size of 174 bp was obtained from M. vitis, but other five non-target nematodes did not have any amplification bands, thus effectively distinguish M. vitis and the other five species, and could specifically detect the M. vitis from mixed populations.  Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile (J₂) and 10^–4 female.  Futhermore, this PCR technique could be used to detect directly M. vitis from soil samples.  The rapid, sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J₂ of M. vitis and the detection of M. vitis in mixed nematode populations and the detection of two J₂s or one male in 0.5 g soil samples, which will provide technical support for the investigation of the occurrence and damage of M. vitis and the formulation of efficient green control strategies.

Potassium (K), an important nutrient element, can improve the stress resistance/tolerance of crops.  The application of K in resisting plant-parasitic nematodes shows that the K treatment can reduce the occurrence of nematode diseases and increase crop yield.  However, data on K₂SO₄ induced rice resistance against the root-knot nematode Meloidogyne graminicola are still lacking.  In this work, K₂SO₄ treatment reduced galls and nematodes in rice plants and delayed the development of nematodes.  Rather than affecting the attractiveness of roots to nematodes and the morphological phenotype of giant cells at feeding sites, such an effect is achieved by rapidly priming hydrogen peroxide (H₂O₂) accumulation and increasing callose deposition.  Meanwhile, galls and nematodes in rice roots were more in the potassium channel OsAKT1 and transporter OsHAK5 gene-deficient plants than in wild-type, while the K₂SO₄-induced resistance showed weaker in the defective plants.  In addition, during the process of nematode infection, the expression of jasmonic acid (JA)/ethylene (ET)/brassinolide (BR) signaling pathway-related genes and pathogenesis-related (PR) genes OsPR1a/OsPR1b was up-regulated in rice after K₂SO₄ treatment.  In conclusion, K₂SO₄ induced rice resistance against M. graminicola.  The mechanism of inducing resistance was to prime the basal defense and required the participation of the K⁺ channel and transporter in rice.  These laid a foundation for further study on the mechanism of rice defense against nematodes and the rational use of potassium fertilizer on improving rice resistance against nematodes in the field.

Soybean cyst nematode (SCN) Heterodera glycines is considered as the major constraint to soybean production.  GmSHMT08 at Rhg4 locus on chromosome 08, encoding a serine hydroxylmethyltransferase, is a major gene underlying resistance against H. glycines in Peking-type soybeans.  However, the molecular mechanism underpinning this resistance is less well characterized, and whether GmSHMT08 could interact with proteins in H. glycines remains unclear.  In this study, yeast two-hybrid screening was conducted using GmSHMT08 as a bait protein, and a fragment of a 70-kDa heat shock protein (HgHSP70) was screened from H. glycines that exhibited interaction with GmSHMT08.  This interaction was verified by both GST pull-down and bimolecular fluorescence complementation assays.  Our finding reveals HgHSP70 could be applied as a potential candidate gene for further exploring the mechanism on GmSHMT08-mediated resistance against SCN H. glycines.

The cereal cyst nematodes (Heterodera avenae, Heterodera filipjevi, Heterodera latipons) are considered to be one of the most important plant parasitic nematodes attacking most cereals and can cause significant crop losses (Sikora 1988).  In China, H. filipjevi (Madzhidov 1981) Stelter, 1984, was first reported from Henan province (Peng et al. 2010) and a few years later in Anhui province and Xinjiang Uygur Autonomous Region (Peng et al. 2016, 2018) .  In December 2017, a survey for cereal cyst nematodes on winter wheat was conducted in Shandong Province, China.  A total of 79 samples that including roots and rhizosphere soil were collected.  Cysts and second-stage juveniles (J2s) were isolated from each soil sample using the sieving-decanting method.  Wheat roots were stained with acid fusion to observe the development of cereal cyst nematodes.  One sample collected from Yangzhuan Village in Huanggang Town, Shan County of Heze City (GPS 34°38´23.10´´N, 116°05´42.95´´E), Shandong Province, was found that the wheat roots were heavily parasitized by cyst nematodes, and most of the nematodes in roots had developed to fourth-stage (J4) in mid-December of 2017.  The morphological and molecular studies of cyst and J2s were carried out to confirm the identification of H. filipjevi in one winter wheat field soil and root sample from Shan County.  The cysts were lemon shaped with prominent vulval cone, brown to black in colour.  Cuticle with irregular zig-zag pattern.  Neck prominent, vulval cone bifenestrate with horseshoe-shaped fenestra, bullae and underbridge strongly developed.  The main morphometrics of cysts (n=8) were length (including neck) (688 to 948 μm, mean=794 μm, standard deviation=87 μm), width (465 to 620 μm, mean=529 μm, standard deviation=63 μm), neck length (71.5 to 126.3 μm, mean=86.5 μm, standard deviation=9.2 μm), fenestra length (43.8 to 71.3 μm, mean=58.0 μm, standard deviation=15.1 μm), fenestra width (19.8 to 32.0 μm, mean=25.0 μm, standard deviation=3.9 μm), length of vulval slit (8.1 to 9.7 μm, mean=9.1 μm, standard deviation=0.5 μm) and length of underbridge (64.5 to 101.3 μm, mean=82.6 μm, standard deviation=12.8 μm).  Measurements of J2s (n=10); body length (556.7 to 617.0 μm, mean=584.3 μm, standard deviation=23.2 μm); stylet (22.8 to 24.1 μm, mean=23.3 μm, standard deviation=0.4 μm), tail (59.6 to 68.6 μm, mean=65.8 μm, standard deviation=3.5 μm) and hyaline tail terminus (35.9 to 41.1 μm, mean=38.6 μm, standard deviation=2.1 μm).  Genomic DNA was isolated from single cysts (n=6), and the internal transcribed spacer regions were amplified with primers TW81 (5´-GTTTCCGTAGGTGAACCTGC-3´) and AB28 (5´-ATATGCTTAAGTTCAGCGGGT-3´) (Joyce et al. 1994) and 28S rDNA-D2/D3 regions were amplified with primers D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and D3B (5´-TCGGAAGGAACCAGCTACTA-3´) (Subbotin et al. 2006).  The obtained internal transcribed spacer regions (ITSs) sequences (GenBank accession MG859977) is 99% identical to those of H. filipjevi from Turkey (KR704292.1 and KR704304.1), the United States (KP878490.1 and GU079654.1) and China (KY448473.1 and KY448473.1).  The obtained 28S rDNA-D2/D3 sequences (GenBank accession MG859980) also to be 99 to 100% identical to those of H. filipjevi from China (GU083597.1, KT314235.1, GU083592.1).  The species-specific primers of H. filipjevi (HfF1, 5´-CAGGACGAAACTCATTCAACCAA-3´; HfR1, 5´-AGGGCGAACAGGAGAAGATTAGA-3´) were also used to identify this population (Peng et al. 2013), the specific band was obtained species-specific primers of H. filipjevi.  Based on the morphological and molecular data, the species of the cyst-forming nematode was identified as H. filipjevi.  As far as we know, this is the first report of H. filipjevi in Shandong Province, China.  The population density of H. filipjevi were found much higher than those of other CCN, it can serious infect winter wheat at seedling stage which often cause economically damaging to wheat, so the spread of H. filipjevi would be a risk for the cereal production of Shandong province. 

The root-knot nematode Meloidogyne graminicola, which is distributed worldwide, is considered a major constraint on rice production in Asia.  The present study used the root gall index and number of nematodes inside the roots to evaluate resistance/susceptibility to M. graminicola in different subpopulations of Oryza sativa (aus, hybrid aus, indica, hybrid indica, temperate japonica, tropical japonica).  Nematode development in highly resistant varieties was also evaluated.  Analyses of randomly selected 35 varieties showed the number of M. graminicola nematodes inside the roots correlated very strongly (r=0.87, P≤0.05) with the nematode gall index, and the results from pot and field experiments revealed similar rankings of the varieties for resistance/susceptibility.  Among the 136 tested varieties, temperate japonica displayed the highest gall index, followed by tropical japonica, indica, hybrid indica, aus, and hybrid aus. Zhonghua 11 (aus), Shenliangyou 1 (hybrid aus) and Cliangyou 4418 (hybrid indica) were highly resistant to M. graminicola under both pot and field conditions.  Further examination of nematode development suggested that compared to susceptible rice, M. graminicola penetrated less often into highly resistant varieties and more frequently failed to develop into females.  The promising varieties found in the present research might be useful for the breeding of hybrid rice in China and for the further development of practical nematode management measures.   

Plant-parasitic nematodes are very common on cereal crops and cause economic losses via reduction in grain quality and quantity. During 2014, 83 soil samples were collected from wheat and barley fields in 21 districts of 13 provinces across five regions (Central Anatolia, Marmara, Aegean, Southeast Anatolia, and Black Sea Region) of Turkey. Cyst-forming nematodes were found in 66 samples (80%), and the internal transcribed spacer (ITS) sequencing and species-specific PCR identified the species in 64 samples as Heterodera filipjevi, Heterodera latipons, and Heterodera avenae. The predominant pathogenic cereal cyst nematode was H. filipjevi, which was found in all five regions surveyed. H. avenae was only detected in Southeast Anatolia whereas H. latipons was detected in Southeast Anatolia and Central Anatolia. ITS-rDNA phylogenetic analyses showed that H. avenae isolates from China clustered with H. australis, and Turkish isolates were closely related to European and USA isolates of this species. H. filipjevi from Turkey and China were clustered closely with those from the UK, Germany, Russia, and the USA. The density of many of these populations exceeded or approached the maximum threshold level for economic loss. To our knowledge, this is the first report of H. filipjevi in Diyarbakir, Edirne, and Kutahya provinces, and the first report of H. avenae in Diyarbakir Province. These results exhibit the most rigorous analysis to date on the occurrence and distribution of Heterodera spp. in Turkey’s major wheat-producing areas, thus providing a basis for more specific resistance breeding, as well as other management practices.

    Heterodera avenae (cereal cyst nematode, CCN) infects many cereal crops and causes serious yield losses worldwide. Interaction studies investigating H. avenae and its hosts are still in their infancy. In this study, a barley model plant, the Hordeum vulgare cultivar Golden Promise, was investigated for its potential as a candidate model host to study its interaction with H. avenae. CCN-infective juveniles were attracted by the root tips and gathered around the root elongation zones of Golden Promise on 0.7% water agar plates. The juveniles invaded the roots and developed successfully until maturation at 40 days after inoculation in sterile sand soil. The cryotomy and syncytium measurements indicated that the syncytia enlarged gradually throughout the development of the nematodes and caused the corresponding root regions to swell obviously. Quantitative real-time PCR analysis showed that the down-regulation of defence-related barley genes and up-regulation of development-related barley genes contribute to the understanding of compatible interaction between H. avenae and Golden Promise. Barley stripe mosaic virus (BSMV) virus-induced gene silencing (VIGS) can be used in the roots of Golden Promise. In conclusion, the Hordeum vulgare cultivar Golden Promise is a suitable candidate model host for interaction studies with Heterodera avenae. The studies presented above document the first CCN host that not only has published genome context but also be compatible to BSMV VIGS.  

Soybean cyst nematode Heterodera glycines is one of the most serious soil-borne pathogens in soybean production. However, the researches were limited in China due to lack of an effective pathosystem. In this study, we screened 21 legume Medicago plants in both Medicago truncatula and Medicago sativa to obtain candidate model plants for establishing a new pathosystem for legume-H. glycines interactions. The nematode infection of tested plants was assayed with Race 3 and 4 respectively, which were two dominant H. glycines inbred races in China soybean producing areas. The results showed that the model legume plant M. truncatula A17 failed to allow Race 3 of H. glycines to complete its life cycle, in contrast, it provided the Race 4 population to form several cyst nematodes, however, the female index (FI) value was approximately 1.6. Three M. sativa cultivars, including Xunlu, Aergangjin and Junren, provided either Race 3 or 4 of H. glycines to develop into mature cysts with their FI value below 5 as well. Our results demonstrated that legume plants in both M. truncatula and M. sativa were not likely to be a model plant for H. glycines because of an extreme high resistance.

The cereal cyst nematode (CCN, Heteroder aavenae) causes serious yield loss on cereal crops, especially wheat, worldwide. Daxing population in Beijing City and Huangyuan population in Qinghai Province, China, are two CCN populations. In this study, the CCN pathotypes of Daxing and Huangyuan populations were characterized by tests on 23 standard “International Test Assortment” with the local species Wenmai 19 as the susceptible control. Tested materials were grouped by three nematode populations’ virulence on resistant genes (Rha1, Rha2, Rha3, Cre1) and nonresistant genes, varieties and lines. Both Daxing and Huangyuan populations were avirulent to Ortolan (Ha1). Barley cvs. Ortolan, Siri, Morocco, Bajo Aragon 1-1, and Martin 403-2 were all resistant to both populations. Cultivars Herta, Harlan 43 and wheat Iskamish-K-2-light were all susceptible to Huangyuan population, all of them, however, were resistant to Daxing population. The other five oats were all resistant to the two tested CCN populations. Except Iskamisch K-2-light, all the other wheat cultivars (Capa, Loros×Koga, AUS 10894, and Psathias) were susceptible to Daxing population. Because the pathotypes of the two tested CCN populations in Beijing and Qinghai were not identical to any of the 13 pathotypes previously characterized by the test assortment, we classified Daxing and Huangyuan populations as the new pathotypes, named Ha91.

For sedentary endo-parasitic nematodes, parasitism genes encoding secretory protein expressed in the subventral glands cells always play an important role during the early parasitic process. A new acid phosphatase gene (Ha-acp1) expressed in the subventral glands of the cereal cyst nematode (Heterodera avenae) was cloned and the characteristics of the gene were analyzed. Results showed that the gene had a putative signal peptide for secretion and in situ hybridization showed that the transcripts of Ha-acp1 accumulated specifically in the subventral gland cells of H. avenae. Southern blot analysis suggested that Ha-acp1 belonged to a multigene family. RT-PCR analysis indicated that this transcription was strong at the pre-parasitic juveniles. Knocking down Ha-acp1 using RNA interference technology could reduce nematode infectivity by 50%, and suppress the development of cyst. Results indicated that Ha-acp1 could play an important role in destroying the defense system of host plants.

Plant viruses pose significant threats to agriculture, with many vectored by insect pests. The entry of viruses and their encoded proteins into the host nucleus is a critical step for promoting some viral replication and enabling systemic infection. Laodelphax striatellus, also known as the small brown planthopper (SBPH), is an efficient vector for rice stripe virus (RSV), one of the most damaging viruses of rice. In this study, we demonstrate that RSV infection induces the expression of genes in both the classical and non-classical nuclear import pathways of SBPH. A gene belonging to the importin β family, importin 5 (LsIPO5), was upregulated by 84% in SBPH midguts infected with RSV. The nuclear localization signal (NLS, ¹⁶⁸YRSPSKKRHKYV¹⁷⁹) is located within the nonstructural protein NS3 directly bound to LsIPO5, thereby facilitating NS3 nuclear entry. Moreover, a RING-type E3 ligase (LsRING) in SBPH, which mediated the ubiquitination of NS3 in the insect vector, enhanced NS3 binding to LsIPO5 and facilitated NS3 perinuclear localization. Combined treatment of SBPH with both dsIPO5 and dsRING significantly reduced RSV loads, highlighting the importance of LsIPO5 and NS3 ubiquitination cooperation in facilitating viral replication. Our findings provide new insights into synergistic molecular mechanisms that govern RSV infection and suggest potential therapeutic targets to control viral transmission through their insect vectors.