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Three of the cereal cyst nematodes, Heterodera avenae, H. filipjevi and H. latipons are considered to be the most economically important cyst nematodes that affect cultivated cereals around the world. H. filipjevi was first detected in China from Xuchang, Henan Province in 2010 (Peng et al. 2010) and now has been recorded in the Central China of Henan, Shandong and Anhui provinces and the Northwest China of Xinjiang Uygur Autonomous Region (Cui et al. 2020). In June 2019, 42 samples consisting of roots and soil were collected from winter wheat fields in Hebei Province of North China. Cysts were detected in 37 soil samples with a mean of 6.4 ± 1.67 cysts per 100 ml of soil. Cysts and second-stage juveniles (J2s) were extracted from root and soil following Cobb's sieving gravity method. Morphological and molecular studies of J2s and cysts confirmed its identity with H. filipjevi in 5 samples from Handan (N36°10'052" and E114°35'056"; N36°37'054" and E114°22'052"), Xingtai (N36°53'060" and E114°30'011") and Shijiazhuang (N 37°26'048" and E 116°05'039") in Hebei Province, China. Morphologically, the cysts are lemon-shaped, light or dark brown in color. The vulval cone is bifenestrate with horseshoe-shaped semifenestrae, strongly globular bullae, and well-developed underbridge. Measurements (mean +_ sd (range)) of cysts (n=10), body length not including neck is 743.0 ± 36.1 μm (665 - 780 μm), body width is 559.0 ± 50.0 μm (455 - 639 μm), length / width ratio is 1.33 ± 0.07 (1.20 - 1.46); neck length is 99.3 ± 8.8 μm (85 - 122 μm); fenestrae length is 56.8 ± 5.0 μm (49 - 65 μm) and width is 25.5 ± 1.8 μm (21.1 - 27.8 μm); underbridge length is 84.0 ± 8.1 μm (62 - 93 μm); and vulval slit length is 8.6 ± 0.5 μm (7.2 - 9.1 μm). Measurements of J2s (n = 12), body length is 541 ± 11.4 μm (490 - 578 μm); stylet length is 22.3 ± 0.5 μm (22.0 - 25.0 μm) with anchor-shaped basal knobs; tail length is 57.7 ± 3.7 μm (52.7 - 65.2 μm), and hyaline tail terminal length is 36.5 ± 2.8 μm (32 - 39.8 μm). The tail had a sharp terminus. Morphology of the cysts and J2s were consistent with the record of H. filipjevi (Peng et al. 2010; Subbotin et al. 2010). The amplifications of rDNA-internal transcribed spacer (ITS) fragments were generated with a PCR fragment of 1054 bp from single cysts of each population, using primers TW81 and AB28 (Joyce et al. 1994). The PCR tests for each sample were repeated five times. The PCR product was purified and sequenced. All nucleotide sequences of ITS-rDNA were submitted to GenBank under accession numbers MW282843-6. Sequences from the ITS region were more than 99.5% identical to those of H. filipjevi from Egypt (KF225725), Turkey (KR704308, KR704293 and MN848333) and China (KT314234, MT254744 and KY448473). These results from ITS supported its identity as H. filipjevi. The results were also confirmed by species specific sequence characterized amplified region primers of H. filipjevi (Peng et al. 2013). Pathogenicity of the H. filipjevi was confirmed by infection of winter wheat (Triticum aestivum L cv. 'Aikang58') and examination of the nematode development and reproduction. Wheat seeds were germinated in petri dishes and then transplanted into five polyvinyl chloride tubs (3 cm in diameter, 25 cm in length) that contained 150 cm3 of a sterile soil mixture (loamy soil: sand = 1:1), each with 5 cysts (mean of 252.0 eggs/cyst). Plants were grown in an artificial climate box for one week at 14/18°C, two weeks at 16/20°C, five weeks at 18/25°C and two weeks at 22/30°C, under 8 h of darkness/16 h light and normal culturing practices (Cui et al. 2015). The parasitic J2s, third and fourth-stage juveniles, and adult females were observed in roots stained with acid fuchsin at 10, 20, 30, and 50 days after inoculation (DAI), and an average of 32.0 cysts per tubes were extracted 70 DAI. The new cyst' morphological and molecular characteristics were identical to the H. filipjevi cysts from the original soil samples. Three other tubes without cysts were set as control and there were no newly formed cysts. Heterodera avenae and H. filipjevi had been detected in a total of 16 wheat-producing provinces in China, which resulted in losses of 1.9 billion CNY year-1 (Cui et al. 2015). To our knowledge, this is the first report of H. filipjevi in Hebei Province of North China. Cereal cyst nematodes are easily transferred to non-infested areas by many avenues, resulting in increased species and pathotype complexity (Cui et al. 2020). Once H. filipjevi continues to spread in main wheat producing area of China, it could become be a new threat to cereals production. It is time to take effective control methods to prevent H. filipjevi further dispersal, especially through the farming machinery transmission. Hebei Province is one of the most important major grain-producing areas, our findings will be very beneficial for H. filipjevi management and further research on winter wheat in Hebei Province, North China.

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, Lo-rosxKoga, 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.

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 northern root-knot nematode (Meloidogyne hapla) is a damaging nematode that has caused serious economic losses worldwide. In the present study, a sensitive, simple and rapid method was developed for detection of M. hapla in infested plant roots by combining a Flinders Technology Associates (FTA) card with loop-mediated isothermal amplification (LAMP). The specific primers of LAMP were designed based on the distinction of internal transcribed spacer (ITS) sequences between M. hapla and other Meloidogyne spp. The LAMP assay can detect nematode genomic DNA at concentrations low to 1/200 000, which is 100 times more sensitive than conventional PCR. The LAMP was able to highly specifically distinguish M. hapla from other closely related nematode species. Furthermore, the advantages of the FTA-LAMP assay to detect M. hapla were demonstrated by assaying infected root galls that were artificially inoculated. In addition, M. hapla was successfully detected from six of forty-two field samples using FTA-LAMP technology. This study was the first to provide a simple diagnostic assay for M. hapla using the LAMP assay combined with FTA technology. In conclusion, the new FTA-LAMP assay has the potential for diagnosing infestation in the field and managing the pathogen M. hapla.

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.

Glyphosate-resistant (GR) transgenic soybean has never been cultivated commercially in China. It is essential to develop the separation measures required to prevent out-crossing between GR and conventional soybean (Glycine max (L.) Merr.) by characterizing the transgene flow before GR soybean is released. In this study, the transgene flow from a GR transgenic soybean AG5601 to conventional soybeans was characterized. First, natural out-crossing rate was evaluated using 36 conventional soybean varieties interplanted with GR soybean AG5601 transformed with a cp4 EPSPS gene conferring the resistance to herbicide glyphosate in the field in 2007 and 2008 in China. Second, drift distance of cp4 EPSPS gene from GR soybean AG5601 to soybean cv. Zhonghuang13 was evaluated using the progenies harvested from eight directions at different distance. Third, the relationship of gene flow of GR soybean AG5601 with flowering synchronization days or insect pollinators of each variety was analyzed using regression analysis. Thirty-two of 36 tested conventional soybean varieties had surviving progenies after two times of sprays of glyphosate, and 49 of 41,679 progenies were verified to be glyphosate-tolerant heterozygous offspring. The out-crossing rates in positive varieties (having surviving offspring after two times of sprays of glyphosate) ranged from 0.039 to 0.934 %. The farthest distance (drift distance) between soybean AG5601 and cv. Zhonghuang13 at which out-pollinating was still able to be observed was 15 m, with an out-crossing rate of 0.012 %. Regression analysis showed that there was a positive relationship between cross-pollination frequency and flowering synchronization days or pollinator insects. Therefore, when GR soybean is released to the field, it should be critically separated with the conventional soybean in space and cultivation time with efficient insect control during flowering.

The root-knot nematode Meloidogyne incognita causes severe damage to continuously cropping vegetables. The control of this nematode relies heavily on organophosphate nematicides in China. Here, we described resistance to the organophosphate nematicide fosthiazate in a greenhouse-collected resistant population (RP) and a laboratory susceptible population (SP) of M. incognita. Fosthiazate was 2.74-fold less toxic to nematodes from RP than that from SP. Quantitative real-time PCR revealed that the acetylcholinesterase2 (ace2) transcription level in the RP was significantly higher than that in the SP. Eighteen nonsynonymous amino acid differences in ace2 were observed between the cDNA fragments of the RP and SP. The acetylcholinesterase (AChE) protein activity in the RP was significantly reduced compared with that in the SP. After knocking down the ace2 gene, the ace2 transcription level was significantly decreased, but no negative impact on the infection of juveniles was observed. The 50% lethal concentration of the RNAi RP population decreased 40%, but the inhibition rate of fosthiazate against AChE activity was significantly increased in RP population. Thus, the increased fosthiazate insensitivity in the M. incognita resistant population was strongly associated with mutations in ace2. These results provide valuable insights into the resistance mechanism of root-knot nematode to organophosphate nematicides.

Cereal cyst nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands and are secreted into plant tissues through the stylet. To understand the function of nematode effectors in parasitic plants, we cloned predicted effectors genes from Heterodera avenae and transiently expressed them in Nicotiana benthamiana. Infiltration assays showed that HaEXPB2, a predicted expansin-like protein, caused cell death in N. benthamiana. In situ hybridization showed that HaEXPB2 transcripts were localised within the subventral gland cells of the pre-parasitic second-stage nematode. HaEXPB2 had the highest expression levels in parasitic second-stage juveniles. Subcellular localization assays revealed that HaEXPB2 could be localized in the plant cell wall after H. avenae infection.This The cell wall localization was likely affected by its N-terminal and C-terminal regions. In addition, we found that HaEXPB2 bound to cellulose and its carbohydrate-binding domain was required for this binding. The infectivity of H. avenae was significantly reduced when HaEXPB2 was knocked down by RNA interference in vitro. This study indicates that HaEXPB2 may play an important role in the parasitism of H. avenae through targeting the host cell wall.

Purpureocillium lilacinum is a promising nematophagous ascomycete able to adapt diverse environments and it is also an opportunistic fungus that infects humans. A microbial inoculant of P. lilacinum has been registered to control plant parasitic nematodes. However, the molecular mechanism of the toxicological processes is still unclear because of the relatively few reports on the subject. In this study, using Illumina paired-end sequencing, the draft genome sequence and the transcriptome of P. lilacinum strain 36-1 infecting nematode-eggs were determined. Whole genome alignment indicated that P. lilacinum 36-1 possessed a more dynamic genome in comparison with P. lilacinum India strain. Moreover, a phylogenetic analysis showed that the P. lilacinum 36-1 had a closer relation to entomophagous fungi. The protein-coding genes in P. lilacinum 36-1 occurred much more frequently than they did in other fungi, which was a result of the depletion of repeat-induced point mutations (RIP). Comparative genome and transcriptome analyses revealed the genes that were involved in pathogenicity, particularly in the recognition, adhesion of nematode-eggs, downstream signal transduction pathways and hydrolase genes. By contrast, certain numbers of cellulose and xylan degradation genes and a lack of polysaccharide lyase genes showed the potential of P. lilacinum 36-1 as an endophyte. Notably, the expression of appressorium-formation and antioxidants-related genes exhibited similar infection patterns in P. lilacinum strain 36-1 to those of the model entomophagous fungi Metarhizium spp. These results uncovered the specific parasitism of P. lilacinum and presented the genes responsible for the infection of nematode-eggs.

The alpha-proteobacteria of the genus Wolbachia is a widespread group of maternally inherited endosymbionts of arthropod and nematode hosts. Wolbachia infection induces a range of host phenotypes, including cytoplasmic incompatibility, male killing, feminization, and induction of thelytokous parthenogenesis. Heterogony (cyclical parthenogenesis) is a remarkable characteristic of oak gallwasps, Cynipini, the largest tribe of the Cynipidae. A few species of Cynipini are exceptional in that they are univoltine and exhibit thelytokous parthenogenesis, probably because they lost the arrhenotokous generation of their heterogonic ancestor species due to Wolbachia infection. In this study, the presence of Wolbachia was detected using polymerase chain reaction primers for the wsp genes in a thelytokous parthenogenetic species [Dryocosmus kuriphilus (Yasumatsu)] (Hymenoptera: Cynipidae: Cynipini). Approximately 29.8 and 87.1% of adults of the Zhuzhou and Fuzhou strains, respectively, were infected with Wolbachia while all females of the remaining four strains collected from other localities in China were Wolbachia free. The length of the wsp fragment of Zhuzhou and Fuzhou strains was found to be 573 and 561 bp, respectively. The nucleotide sequence of the bacterial wsp fragment indicated that the endosymbiotic bacteria of the Zhuzhou and Fuzhou strains are members of supergroup A, but belong to different clades; they probably originated from two independent infection events. In conclusion, thelytokous parthenogenesis of D. kuriphilus is not caused by Wolbachia infection and the deletion of the arrhenotokous generation is thus not associated with such an infection.

Background Cereal cyst nematode Heterodera avenae, an important soil-borne pathogen in wheat, causes numerous annual yield losses worldwide, and use of resistant cultivars is the best strategy for control. However, target genes are not readily available for breeding resistant cultivars. Therefore, comparative transcriptomic analyses were performed to identify more applicable resistance genes for cultivar breeding. Methods The developing nematodes within roots were stained with acid fuchsin solution. Transcriptome assemblies and redundancy filteration were obtained by Trinity, TGI Clustering Tool and BLASTN, respectively. Gene Ontology annotation was yielded by Blast2GO program, and metabolic pathways of transcripts were analyzed by Path_finder. The ROS levels were determined by luminol-chemiluminescence assay. The transcriptional gene expression profiles were obtained by quantitative RT-PCR. Results The RNA-sequencing was performed using an incompatible wheat cultivar VP1620 and a compatible control cultivar WEN19 infected with H. avenae at 24 h, 3 d and 8 d. Infection assays showed that VP1620 failed to block penetration of H. avenae but disturbed the transition of developmental stages, leading to a significant reduction in cyst formation. Two types of expression profiles were established to predict candidate resistance genes after developing a novel strategy to generate clean RNA-seq data by removing the transcripts of H. avenae within the raw data before assembly. Using the uncoordinated expression profiles with transcript abundance as a standard, 424 candidate resistance genes were identified, including 302 overlapping genes and 122 VP1620-specific genes. Genes with similar expression patterns were further classified according to the scales of changed transcript abundances, and 182 genes were rescued as supplementary candidate resistance genes. Functional characterizations revealed that diverse defense-related pathways were responsible for wheat resistance against H. avenae. Moreover, phospholipase was involved in many defense-related pathways and localized in the connection position. Furthermore, strong bursts of reactive oxygen species (ROS) within VP1620 roots infected with H. avenae were induced at 24 h and 3 d, and eight ROS-producing genes were significantly upregulated, including three class III peroxidase and five lipoxygenase genes. Conclusions Large-scale identification of wheat resistance genes were processed by comparative transcriptomic analysis. Functional characterization showed that phospholipases associated with ROS production played vital roles in early defense responses to H. avenae via involvement in diverse defense-related pathways as a hub switch. This study is the first to investigate the early defense responses of wheat against H. avenae, not only provides applicable candidate resistance genes for breeding novel wheat cultivars, but also enables a better understanding of the defense mechanisms of wheat against H. avenae. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2037-8) contains supplementary material, which is available to authorized users.

Lecanicillium lecanii has been developed as a biopesticide and used in biological control of several agricultural insects. To improve fungal virulence, an optimised polyethylene glycol (PEG)-mediated protoplast transformation system was established for L. lecanii. Pr1A-like cuticle-degrading protease gene (Cdep1) from Beauveria bassiana was transferred into L. lecanii, and its resulting activity against Aphis gossypii was assessed.The optimised protoplast generation yielded 2.5 × 10(8) protoplasts g(-1) wet mycelium of fungi, and gave nearly 98% viability and 80% regeneration on plates. Protease activities were increased about fivefold in transformants expressing CDEP1. The median lethal concentration (LC50 ) for transformants expressing CDEP1 was twofold lower than that for the wild type (WT). The median survival time (LT50 ) for transformants expressing CDEP1 was also 14.2% shorter than that for WT, though no significant difference. There were no significant differences in conidial germination as colony growth and conidial yield on plates between transformants expressing CDEP1 and WT. The transformants expressing CDEP1 grew significantly quicker than WT in insects. The transformants expressing CDEP1 were lower in conidial yields on insect cadavers, but insignificantly different from WT.The PEG-mediated protoplast transformation system was effective for L. lecanii, and the expression of CDEP1 significantly enhanced fungal virulence against cotton aphids. © 2016 Society of Chemical Industry.

Alternative splicing (AS) is common in higher eukaryotes and plays an important role in gene posttranscriptional regulation. It has been suggested that AS varies dramatically among species, tissues, and duplicated gene families of different sizes. However, the genomic forces that govern AS variation remain poorly understood. Here, through genome-wide identification of AS events in the soybean (Glycine max) genome using high-throughput RNA sequencing of 28 samples from different developmental stages, we found that more than 63% of multiexonic genes underwent AS. More AS events occurred in the younger developmental stages than in the older developmental stages for the same type of tissue, and the four main AS types, exon skipping, intron retention, alternative donor sites, and alternative acceptor sites, exhibited different characteristics. Global computational analysis demonstrated that the variations of AS frequency and AS types were significantly correlated with the changes of gene features and gene transcriptional level. Further investigation suggested that the decrease of AS within the genome-wide duplicated genes were due to the diminution of intron length, exon number, and transcriptional level. Altogether, our study revealed that a large number of genes were alternatively spliced in the soybean genome and that variations in gene structure and transcriptional level may play important roles in regulating AS.