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Summary For efficient use of available synthesised nematicides, bioassay-guided analysis is required to understand the behaviours of nematicides. In this study, the key nematicides on the market, including avermectin B1a, avermectin B2a, emamectin benzoate, fluensulfone, fluopyram and fosthiazate, were evaluated by both in vitro and in planta approaches with optimised solvent and nematode inoculum. We found that acetone was the only one of the six solvents tested safe for both root-knot nematode (RKN) second-stage juveniles (J2) and cucumber at the tested concentration. The optimal numbers of RKN J2 for inoculation on cucumber seedlings in the glass-tube assay, cup assay and microplot experiment were 200, 700 and 1250, respectively. No obvious phytotoxicity symptoms were observed in the in planta assays for all nematicides selected at the tested concentration in the soil drench application. When the substrate changed from pure sand to matrix-sand, the EC50 and EC90 of all six nematicides increased. Among them, fluopyram rose the most. In the microplot experiment, avermectin B2a showed the lowest EC50 but the EC90 of avermectin B2a, fluensulfone, fluopyram and fosthiazate were similar. The results are useful as a guide to the application of commonly used nematicides in the field.

Insect TRPV is one subfamily of transient receptor potential (TRP) channels, and it is composed of Inactive (Iav) and Nanchung (Nan), the molecular targets of afidopyropen in several sucking insect pests. In this study, we performed successive selection and synergism tests based on previous work. The resistant afidopyropen strain HD-Afi of Bemisia tabaci reached about 86-fold resistance to afidopyropen, and only part of the resistance resulted from detoxification of cytochrome P450 monooxygenases. Then, we cloned and characterized Nan and Iav from B. tabaci (BtNan and BtIav), and found that they were expressed in all stages of development and tissues of adult, and the expression level of BtNan in strains of HD-S and HD-Afi were significantly increased after afidopyropen treatment. Further, expression of BtNan was downregulated after gene silencing, and it resulted in significantly decreased afidopyropen resistance in the strain of HD-Afi. Our data revealed the first evidence that overexpression of the TRPV Nan gene is responsible for causing afidopyropen resistance in B. tabaci, and our results could provide new visions on the function of TRPVs in the development of resistance to pesticide and supply evidence for developing novel strategies of pest management.

To investigate the influence of the pyrolysis temperature on biogas production from sewage sludge, conventional pyrolysis and microwave pyrolysis were carried out in the temperature range from 600 °C to 900 °C and all products were analyzed. With the temperature increasing, the product yields for conventional pyrolysis varied significantly, while those for microwave pyrolysis changed quite slightly. In conventional pyrolysis, the yield of H2 increased from 1.26 mmol/g at 600 °C to 9.07 mmol/g at 900 °C, while it was varied only from 1.84 mmol/g to 3.67 mmol/g in microwave pyrolysis. Under microwave pyrolysis, a high ratio of H/C indicated that more hydrogen atoms converted directly to tar instead of being released into biogas, which was caused by side reactions (such as the hydrogen transfer reaction). More aromatic compounds in the tar during microwave pyrolysis illustrated that the hydrogen transfer reaction was enhanced by microwave at the higher temperatures. It has been found that the sludge microwave pyrolysis had some drawbacks for the hydrogen-rich biogas production, because it could promote some side reactions to suppress the H2 production, especially the hydrogen transfer reaction.

Acylsugars are secondary metabolites that are produced in the trichomes of some solanaceous species and can help control several herbivorous insect pests. Previously, knockout mutations (

We investigated the strength and deformation characteristics of undisturbed unsaturated loess under different moisture content conditions. The results showed that the deviatoric stress intensity increases with the increase of confining pressure at the same moisture content and decreases with increasing moisture content at the same confining pressure. Moreover, the matric suction and the shear strength of unsaturated loess gradually decrease as the moisture content increases. It was found that cohesion in unsaturated loess is significantly larger than that in saturated soil. Under the same moisture content, the volumetric strain decreases as the axial strain increases, but this effect is less significant as the confining pressure increases. Moreover, at the same confining pressure, the volumetric strain increases with the moisture content. Based on the theory of unsaturated soil strength, the increasing gradient of cohesion, internal friction angle, and the relationship between the shear strength and the matric suction of the corresponding soil layers were obtained. These findings provide a reliable theoretical basis for the optimal design of tunnel linings, the selection of appropriate construction methods, and the evaluation of the stability of tunnel soils during construction.

Background Pair-rule genes were identified and named for their role in segmentation in animal embryos. Paired, a homolog of mammalian PAX3, acts as one of several pair-rule genes and is key in defining the boundaries of future parasegments and segments during insect embryogenesis. Results We cloned the paired gene from the oriental fruit fly, Bactrocera dorsalis, and then applied CRISPR/Cas9-mediated genome editing to investigate its physiological function in the embryonic stage of this pest. We identified one transcript for a paired homolog in B. dorsalis, which encodes a protein containing a Paired Box domain and a Homeobox domain. Phylogenetic analysis confirmed that the paired gene is highly conserved and the gene was highly expressed at the 12-14 h-old embryonic stage. Knock-out of Bdpaired led to lack of segment boundaries, cuticular deficiency, and embryonic lethality. Sequence analysis of the CRISPR/Cas9 mutants exhibited different insertion and deletions in the Bdpaired locus. In addition, the relative expression of Wingless (Wg) and Abdominal A (Abd-A) genes were significantly down-regulated in the Bdpaired mutant embryos. Conclusion These results indicate that Bdpaired gene is critical for the embryonic development of B. dorsalis, and could be a novel molecular target for genetic-based pest management practices to combat this serious invasive pest. © 2019 Society of Chemical Industry.

Block stability analysis is important for studying the overall stability of surrounding rock in slope engineering. The main problem in regards to block stability analyses is determining the relative positional relationship between the structural plane and the free face. Consequently, studying the structural plane and free face is of great significance in engineering. By using the steep rocky slope of Shenxianju in Xianju County, Zhejiang Province as an example, this paper proposes new methods for determining the structural plane and free face using the coordinate projection method through an unmanned aerial vehicle. In addition, using MATLAB, the authors developed a CPG programme suitable for rock slope engineering and established a three-dimensional (3D) model of the slope allowing the visualisation of the structural plane, free face and slope characteristics. This practical engineering application demonstrates that this method is reliable and that the CPG programme is feasible. Overall, this study provides new technical means for landslide prediction and prevention and proposes an effective solution for efficient and safe operation in such instances.

The Heidong Cavern Group of China includes 21 large-scale caverns that have remained stable for 1400 years. Cavern No. 5 of the Heidong Cavern Group is characterized by an unsupported span of up to 92 m, which is considered to be the largest span in a single underground artificial rock cavern. Cavern No. 5 may have gone through a period during which it was the only cavern and a period during which it had adjacent caverns; recent investigations have proved that Cavern No. 5 is suffering from considerably serious partial damage. The factors affecting the stability of Cavern No. 5, including waste rock accumulations, rock mass structures, surrounding rock quality, and cavern structures, were analyzed to prevent further damage and analyze the influence of the existence of adjacent caverns on the stability of Cavern No. 5. The numerical analysis software MIDAS GTS (Geotechnical and tunnel analysis system) was used to simulate and compare the stability of Cavern No. 5 under the single-cavern and adjacent-cavern cases. Research proves that when this cavern is independently considered and the remaining caverns are excluded, the arched roof structure is considerably favorable to ensure the cavern’s stability and the horizontal compressive stress of the roof becomes approximately equivalent to the vertical tensile stress. The horizontal forces generated by the cavern’s inclined roof contribute to the cavern’s stability. The horizontal compressive stress area of the roof and the effect of the arched structure decrease when adjacent caverns are present, causing the roof to become prone to tensile failure. These results provide a reference for modern underground engineering construction and a theoretical basis for long-term protection of the large and ancient Heidong Caverns.

Organic pollutants are widespread environmental pollutants with high toxicity, persistence, and bioaccumulation. Our aim was to investigate the distribution of aromatic amines, phenols, chlorobenzenes, and naphthalenes in the surface sediment of the Dianchi Lake, China. Nineteen surface sediment samples were collected from the Dianchi Lake, and 40 types of organic pollutants were analyzed via gas chromatography–mass spectrometry. The total organic pollutant concentrations in the surface sediment of the Dianchi Lake varied from 27.4 to 1.62 × 103 ng/g. The concentrations of phenols were much higher than those in other water bodies but still within a controllable range, whereas the concentrations of the other organic pollutant classes were similar or even lower. The detection ratio of 3- or 4-methylphenol was the highest (100.00%) among the pollutants. The average total organic pollutant concentrations decreased in the following order: Caohai (540 ng/g)>the middle of Waihai (488 ng/g)>the edge of Waihai (351 ng/g)>Haigeng Dam (90.4 ng/g). Pearson analysis showed a strong correlation among 1-methylnaphthalene, 2-methylnaphthalene, 1,3-dinitronaphthalene, and 1,4-dinitronaphthalene (p

To expand the usage of endophytes in agriculture and in forestry, the insecticidal gene cry218 of Bacillus thuringiensis was introduced into a poplar bacterial endophyte Burkholderia pyrrocinia JK-SH007. The cry218 gene was cloned by polymerase chain reaction (PCR) and was inserted into a PHKT2 expression vector that was introduced into the bacterial endophyte JK-SH007. By using sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE) and western blotting, we confirmed that the engineered bacterial endophyte was successfully constructed, and it harboured insecticidal function after the bioassay in planta. The toxicity of the expressed insecticidal protein was analysed on second instar silkworm. The regression equation showed that the median lethal concentration (LC50) of the insecticidal protein was 0.77 (0.57–1.04) g/L at 72 h. The insecticidal bacteria genetically modified in this study have laid the foundation for further exploitation of biocontrol bacteria.

Background The gut bacteria of tephritid fruit flies play prominent roles in nutrition, reproduction, maintenance and ecological adaptations of the host. Here, we adopted an approach based on direct observation of symbiotic or axenic flies feeding on dishes seeded with drops of full diet (containing all amino acids) or full diet supplemented with bacteria at similar concentrations to explore the effects of intestinal bacteria on foraging decision and fitness of Bactrocera dorsalis. Results The results show that intestinal probiotics elicit beneficial foraging decision and enhance the female reproduction fitness and survival of B. dorsalis (symbiotic and axenic), yet preferences for probiotic diets were significantly higher in axenic flies to which they responded faster compared to full diet. Moreover, females fed diet supplemented with Pantoea dispersa and Enterobacter cloacae laid more eggs but had shorter lifespan while female fed Enterococcus faecalis and Klebsiella oxytoca enriched diets lived longer but had lower fecundity compared to the positive control. Conversely, flies fed sugar diet (negative control) were not able to produce eggs, but lived longer than those from the positive control. Conclusions These results suggest that intestinal bacteria can drive the foraging decision in a way which promotes the reproduction and survival of B. dorsalis. Our data highlight the potentials of gut bacterial isolates to control the foraging behavior of the fly and empower the sterile insect technique (SIT) program through the mass rearing.

Background The gut bacteria of tephritid fruit flies play prominent roles in nutrition, reproduction, maintenance and ecological adaptations of the host. Here, we adopted an approach based on direct observation of symbiotic or axenic flies feeding on dishes seeded with drops of full diet (containing all amino acids) or full diet supplemented with bacteria at similar concentrations to explore the effects of intestinal bacteria on foraging decision and fitness of Bactrocera dorsalis. Results The results showed that intestinal probiotics motivate beneficial foraging decision and enhance the female reproduction fitness and survival of B. dorsalis (symbiotic and axenic), yet preferences for probiotic diets were significantly higher in axenic flies to which they responded faster compared to full diet. Moreover, females fed diet supplemented with Pantoea dispersa and Enterobacter cloacae laid more eggs but had shorter lifespan while female fed Enterococcus faecalis and Klebsiella oxytoca enriched diets lived longer but had lower fecundity compared to positive control. Conversely, flies fed sugar diet (negative control) were not able to produce any eggs but lived longer than those from the positive control. Conclusions These results suggest that intestinal bacteria can shape profitable foraging decision in a way which promotes the reproduction and survival of B. dorsalis. Our data could empower the sterile insect technique (SIT) through the mass rearing program.

Scanning ion conductance microscopy (SICM) is an emerging technique for non-contact, high-resolution topography imaging, especially suitable for live cells investigation in a physiological environment. Despite its rapid development, the extended acquisition time issues of its typical hopping/backstep scanning mode still restrict its application for more fields. Herein, we propose a novel SICM scanning approach to effectively reduce the retract distance of existing hopping/backstep mode. In this approach, the SICM probe first gradually descends in the z-direction. Then by using Archimedes spiral trajectory, which has the advantage of higher angular velocity due to its continuous and smooth trajectory, the probe rapidly detects the highest point of the sample in the xy-plane in a layer-by-layer way. Further, the maximum height that decides the retrace distance of pipet in the detected region can be quickly achieved, avoiding a huge retrace distance usually adopted in the existing methods without any prior knowledge (sample height and steepness in the scanning region). Therefore, this new scanning method can greatly reduce the imaging time by minimizing the retrace height of each measurement point. Theoretical analysis is conducted to compare the imaging time of traditional and new method. And various factors in the new method that affect the imaging speed are analyzed. In addition, PDMS (polydimethylsiloxane) and biological samples (C2C12 cells) were imaged by SICM that was operated in the hopping mode, raster-based detecting and developed method with a single-barrel pipet, respectively. The experimental results suggest that the new method has a faster imaging speed than conventional scanning modes but does not sacrifice the imaging quality.

Bioelectrochemical remediation is an emerging technology for in situ removal of petroleum hydrocarbons in soil. Here we demonstrated that the remediation can be extended to a larger range by adding multilayer anodes in contaminated soils. Using a three anodes system with activated carbon as the cathodic catalyst, 918 C of charge transferred during 180 days in aged saline soil. The degradation of both polycyclic aromatic hydrocarbons (PAHs) and n-alkanes were accelerated in each layer compared to the disconnected control. The net degradation rates of total petroleum hydrocarbons, 16 priority PAHs and total n-alkanes (C8–C40) were 18%, 36% and 29%, respectively. Popular exoelectrogens (such as Geobacteraceae sp.) and Escherichia were identified, which possibly played an important role in this bioelectrochemical process.

Secretory proteins encoded by genes expressed in the pharyngeal gland cells of plant-parasitic nematodes have key roles in nematode parasitism of plants. A new β-1,4-endoglucanase gene (designated Ha-eng-1a) was isolated from the cereal cyst nematode Heterodera avenae. The cDNA of Ha-eng-1a encoded a deduced 463-amino acid sequence containing a catalytic domain and a cellulose binding module separated by a linker. The genomic DNA of Ha-eng-1a is 2,129-bp long, containing eight introns ranging from 56 bp to157 bp and nine exons ranging from 70 bp to 299 bp. Southern blot analysis revealed that the Ha-eng-1a gene has two copies. In situ hybridization showed that the Ha-eng-1a transcriptsspecifically accumulated in the two subventral gland cells of the second-stage juveniles. There was evidence for cellulase activity of the recombinant protein Ha-eng-1a in vitro. The results indicated that this β-1,4-endoglucanase gene may play a crucial role in plant cell wall-degradation during penetration and migration of nematodes in the host roots.

SUMMARY The Hg-SYV46 parasitism gene is expressed exclusively in the dorsal oesophageal gland cell of parasitic stages of the soybean cyst nematode, Heterodera glycines , and it encodes a secretory protein that contains a C-terminal motif of the CLAVATA3/ESR-related (CLE) family in Arabidopsis thaliana . In shoot and floral meristems of Arabidopsis, the stem cells secret CLV3, a founding member of the CLE protein family, that activates the CLV1/CLV2 receptor complex and negatively regulates WUSCHEL expression to restrict the size of the stem cell population. Mis-expression of Hg-SYV46 in Arabidopsis (ecotype Columbia-0) under control of the CaMV35S promoter resulted in a wus -like phenotype including premature termination of the shoot apical meristem and the development of flowers lacking the central gynoecium. The wus -like phenotype observed was similar to reports of over-expression of CLV3 and CLE40 in Arabidopsis, as was down-regulation of WUS expression in the shoot apices of 35S::Hg-SYV46/Col-0 plants. Expression of 35S::Hg-SYV46 in a clv31 mutant of Arabidopsis was able partially or fully to rescue the mutant phenotype, probably dependent upon localization and level of transgene expression. A short root phenotype, as reported for overexpression of CLV3 , CLE40 and CLE19 in roots, was also produced in primary 35S::Hg-SYV46/Col-0 transgenic plants. The results suggest a functional similarity of HG-SYV46 to plant-secreted CLE ligands that may play a role in the differentiation or division of feeding cells induced in plant roots by parasitic nematodes.

Secretory proteins encoded by 'parasitism genes' expressed in the oesophageal gland cells of plant-parasitic nematodes play key roles in nematode infection and parasitism of host plants. A cellulose-binding protein-encoding cDNA, designated Hg-cbp-1, was cloned from a Heterodera glycines oesophageal gland-cell long-distance PCR cDNA library. The cDNA hybridised to genomic DNA of H. glycines in Southern blots, and the genomic sequence of Hg-cbp-1 contained only one intron. The Hg-cbp-1 cDNA contained an open reading frame encoding 132 amino acids, with a predicted signal peptide sequence for secretion and a cellulose-binding domain. Bacterial expressed recombinant HG-CBP-1, minus the signal peptide sequence, had no hydrolytic activity on carboxymethyl-cellulose but was able to bind to cellulose. The developmental expression of Hg-cbp-1, determined by real-time reverse transcriptase PCR, showed that Hg-cbp-1 is expressed throughout the parasitic cycle of H. glycines, with a relatively higher expression level in developing parasitic stages.

Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.

A chitinase full-length cDNA (designated Hg-chi-1) was isolated from a Heterodera glycines oesophageal gland cell-specific long-distance PCR cDNA library. The cDNA hybridised to genomic DNA of H. glycines in Southern blots. The Hg-chi-1 cDNA contained an open reading frame encoding 350 amino acids with the first 23 amino acids being a putative signal peptide for secretion. Hg-CHI-1 contained a chitinase 18 family catalytic domain, and chitinolytic activity of recombinant Hg-CHI-1 was confirmed in glycol-chitin substrate gel electrophoresis. In situ mRNA hybridisation analyses showed that transcripts of Hg-chi-1 accumulated specifically in the subventral oesophageal gland cells of parasitic stages of H. glycines, but Hg-chi-1 expression was not detected in eggs or hatched pre-parasitic second-stage juveniles, suggesting that this chitinase does not have a role in egg hatching of H. glycines. The biological function of Hg-CHI-1 in H. glycines remains to be determined.

Secretory proteins encoded by genes expressed in the oesophageal gland cells of plant-parasitic nematodes have key roles in nematode parasitism of plants. Two venom allergen-like protein cDNAs (designated hg-vap-1 and hg-vap-2)were isolated from Heterodera glycines gland cell cDNA libraries. Both cDNAs hybridised to genomic DNA of H. glycines in Southern blots. The hg-vap-1 cDNA contained an open reading frame encoding 215 amino acids with the first 25 amino acids being a putative secretion signal. The hg-vap-2 cDNA contained an open reading frame encoding 212 amino acids with the first 19 amino acids being a putative secretion signal. Genes of hg-vap-1 and hg-vap-2 contained four introns, which ranged in size from 44 to 574 bp, and five exons ranging in size from 43 to 279 bp. In situ hybridisation analyses showed that mRNAs of both vap genes accumulated specifically in the subventral gland cells of H. glycines during parasitism. The gland cell-specific expression and presence of predicted secretion signal peptides in both VAPs suggest that these proteins are secreted from the nematode and may play a role in the infection of host plants by this parasite.

The soybean cyst nematode, Heterodera glycines, is an important pest of soybeans. Although resistance is available against this nematode, selection for virulent races can occur, allowing the nematode to overcome the resistance of cultivars. There are abundant field populations, however, little is known about their genetic diversity. In order to elucidate the differences between races, we investigated the transcriptional diversity within race 3 and race 4 inbred lines during their compatible interactions with the soybean host Zhonghuang 13. Six different race-enriched cDNA libraries were constructed with limited nematode samples collected from the three sedentary stages, parasitic J2, J3 and J4 female, respectively. Among 689 putative race-enriched genes isolated from the six libraries with functional annotations, 92 were validated by quantitative RT-PCR (qRT-PCR), including eight putative effector encoding genes. Further race-enriched genes were validated within race 3 and race 4 during development in soybean roots. Gene Ontology (GO) analysis of all the race-enriched genes at J3 and J4 female stages showed that most of them functioned in metabolic processes. Relative transcript level analysis of 13 selected race-enriched genes at four developmental stages showed that the differences in their expression abundance took place at either one or more developmental stages. This is the first investigation into the transcript diversity of H. glycines races throughout their sedentary stages, increasing the understanding of the genetic diversity of H. glycines.

SUMMARY The soybean cyst nematode, Heterodera glycines, produces beta-1,4-endoglucanases (cellulases) that are secreted during infection of soybean. The gene structures of three, hg-eng-4, hg-eng-5 and hg-eng-6, of the six beta-1,4-endoglucanase genes, all family 5 glycosyl hydrolases previously identified from H. glycines, are presented here. Furthermore, we present the detailed expression analyses of beta-1,4-endoglucanase genes as well as the biochemical properties of four H. glycines endoglucanase enzymes. Two of the endoglucanases, HG-ENG-5 and HG-ENG-6, differed significantly in their amino acid sequence of the catalytic domains and their gene structure from that of the other four beta-1,4-endoglucanases. Quantitative real-time RT-PCR revealed distinct developmental expression differences among the hg-eng family members during the early stages of parasitism and relatively low expression levels in late parasitic stages, with the exception of the adult male stage for some eng genes. Recombinant HG-ENGs degraded carboxymethylcellulose and optimum enzyme activity ranged from pH 5.5 for HG-ENG-5 to pH 8 for HG-ENG-6. EDTA, Ca(2+), Co(2+), Mg(2+) and Fe(2+) did not affect enzyme activity of any ENG protein, whereas Zn(2+), Cu(2+) and Mn(2+) inhibited enzyme activity from 23% to 73% in some cases. In tests with 12 different polysaccharide substrates, enzyme activity was restricted to beta-1,4 linkages with all ENG proteins tested. Only HG-ENG-5 and HG-ENG-6 had relatively high activity on xylan and slightly degraded microcrystalline cellulose. Together, these data reveal distinct differences in expression and biochemistry of cyst nematode parasitism genes and proteins, respectively, and cast light on the intricate interactions between a parasitic animal and its plant host.

Identifying parasitism genes encoding proteins secreted from a nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Meloidogyne incognita parasitism genes were cloned by microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. Of 2,452 cDNA clones sequenced, deduced protein sequences of 185 cDNAs had a signal peptide for secretion and, thus, could have a role in root-knot nematode parasitism of plants. High-throughput in situ hybridization with cDNA clones encoding signal peptides resulted in probes of 37 unique clones specifically hybridizing to transcripts accumulating within the subventral (13 clones) or dorsal (24 clones) esophageal gland cells of M. incognita. In BLASTP analyses, 73% of the predicted proteins were novel proteins. Those with similarities to known proteins included a pectate lyase, acid phosphatase, and hypothetical proteins from other organisms. Our cell-specific analysis of genes encoding secretory proteins provided, for the first time, a profile of putative parasitism genes expressed in the M. incognita esophageal gland cells throughout the parasitic cycle.

Cloning parasitism genes encoding secretory proteins expressed in the esophageal gland cells is the key to understanding the molecular basis of nematode parasitism of plants. Suppression subtractive hybridization (SSH) with the microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences from a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA library. Deduced proteins of 10 clones were preceded by a signal peptide for secretion, and PSORT II computer analysis predicted eight proteins as extracellular, one as nuclear, and one as plasmalemma localized. In situ hybridization showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine in H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cells and the dorsal gland cell, respectively. SSH is an efficient method for cloning putative parasitism genes encoding esophageal gland cell secretory proteins that may have a role in H. glycines parasitism of soybean.

The potato rot nematode, Ditylenchus destructor, is a very destructive nematode pest on many agriculturally important crops worldwide, but the molecular characterization of its parasitism of plant has been limited. The effectors involved in nematode parasitism of plant for several sedentary endo-parasitic nematodes such as Heterodera glycines, Globodera rostochiensis and Meloidogyne incognita have been identified and extensively studied over the past two decades. Ditylenchus destructor, as a migratory plant parasitic nematode, has different feeding behavior, life cycle and host response. Comparing the transcriptome and parasitome among different types of plant-parasitic nematodes is the way to understand more fully the parasitic mechanism of plant nematodes. We undertook the approach of sequencing expressed sequence tags (ESTs) derived from a mixed stage cDNA library of D. destructor. This is the first study of D. destructor ESTs. A total of 9800 ESTs were grouped into 5008 clusters including 3606 singletons and 1402 multi-member contigs, representing a catalog of D. destructor genes. Implementing a bioinformatics' workflow, we found 1391 clusters have no match in the available gene database; 31 clusters only have similarities to genes identified from D. africanus, the most closely related species to D. destructor; 1991 clusters were annotated using Gene Ontology (GO); 1550 clusters were assigned enzyme commission (EC) numbers; and 1211 clusters were mapped to 181 KEGG biochemical pathways. 22 ESTs had similarities to reported nematode effectors. Interestedly, most of the effectors identified in this study are involved in host cell wall degradation or modification, such as 1,4-beta-glucanse, 1,3-beta-glucanse, pectate lyase, chitinases and expansin, or host defense suppression such as calreticulin, annexin and venom allergen-like protein. This result implies that the migratory plant-parasitic nematode D. destructor secrets similar effectors to those of sedentary plant nematodes. Finally we further characterized the two D. destructor expansin proteins.