Репродуктивный потенциал карантинного вредителя сои — соевой нематоды Heterodera glycines в условиях Приморского края

<i>Heterodera glycines</i>

To gain new insights into the mechanism of soybean (Glycine max) interaction with the soybean cyst nematode (Heterodera glycines), we compared protein abundance in soybean roots infected by the soybean cyst nematode at different time intervals. Proteins were extracted from roots of 3 and 8 days post-inoculation (dpi) by soybean cyst nematode Heterodera glycines and separated by 2-DE. A difference in abundance was found in a total of 42 protein spots between the susceptible and resistance responses. After in-gel digestion with trypsin, 39 spots representing 33 proteins were subsequently identified by LC-MS/MS. The proteins showing 1.5-fold changes in intensities are related to biochemical processes that may be differentially altered after soybean cyst nematode challenge. The identified proteins belong to the categories of metabolism, energy, cell growth and division, transcription, protein synthesis, protein destination and storage, signal transduction, disease/defense, and secondary metabolism. Taken together, our study provides important information on the use of proteomic methods for studying protein regulation during soybean-soybean cyst nematode interactions.

The effects of the application of bean sprout residue to soil on the soybean cyst nematode (SCN) Heterodera glycines were evaluated in pot experiments. When bean sprout residue was applied into a soil infested with SCN at a rate of 1%, the number of SCN second-stage juveniles (J2), measured with the Baermann method, increased after 14 days of application and then decreased markedly after 35 days, indicating that some J2 hatched in response to a stimulant in the bean sprout residue and then starved to death in the absence of a host. Next, bean sprout residue was applied two or four times and the number of SCN after 7 weeks was estimated with real-time PCR, which enables quantification of all the forms of SCN (eggs, J2, cysts). Results showed that the SCN density did not change in the untreated control, while it decreased by more than 70% in the residue treatment. Such a hatching-stimulatory and subsequent eradication effect was observed in the water extract of bean sprout residue. Among different parts of bean sprout, both of the stem and root parts had a higher stimulatory effect for hatching, while a stimulatory effect decreased in the stem part greened by culturing under light conditions. These results revealed that bean sprout residue might function as an environmental-friendly control measure for SCN.

Summary Seed germination pouches or seedling growth pouches have been utilised previously for high throughput resistance screening of root-knot nematodes, Meloidogyne spp., on legume plants. In this study we demonstrated that soybean cyst nematode (SCN) Heterodera glycines, with a different reproduction mode from root-knot nematode, is also able to invade, complete its life cycle and reproduce in soybean roots in seedling pouches similar to the pot system with soil, indicating seedling pouches can be utilised as an alternate SCN culture method when glasshouse/growth chamber space is limited. Moreover, seedling pouches provide an easy approach to collect large numbers of clean male nematodes, which could accelerate SCN biological study such as SCN mating behaviour and molecular signals associated with mating. The results of SCN resistance screening indicate that seedling pouches could be utilised for initial resistance screening with SCN.

Soybean cyst nematode (SCN) Heterodera glycines is an obligate parasite that relies on the secretion of effector proteins to manipulate host cellular processes that favor the formation of a feeding site within host roots to ensure its survival. The sequence complexity and co-evolutionary forces acting upon these effectors remain unknown. Here we generated a de novo transcriptome assembly representing the early life stages of SCN in both a compatible and an incompatible host interaction to facilitate global effector mining efforts in the absence of an available annotated SCN genome. We then employed a dual effector prediction strategy coupling a newly developed nematode effector prediction tool, N-Preffector, with a traditional secreted protein prediction pipeline to uncover a suite of novel effector candidates. Our analysis distinguished between effectors that co-evolve with the host genotype and those conserved by the pathogen to maintain a core function in parasitism and demonstrated that alternative splicing is one mechanism used to diversify the effector pool. In addition, we confirmed the presence of viral and microbial inhabitants with molecular sequence information. This transcriptome represents the most comprehensive whole-nematode sequence currently available for SCN and can be used as a tool for annotation of expected genome assemblies.

The primers SCN44f and SCN124r were designed for the quantitative detection of the soybean cyst nematode (SCN) Heterodera glycines using real-time PCR. An andosol naturally infested with SCN was put into a 100 ml core and compacted to various degrees, i.e. 0.93 to 1.4 g cm-3, using a manually-operated compactor. Then, DNA was extracted from the compacted soils as well as non-compacted soil (0.66 g cm-3) and used as templates for real-time PCR. Ct values were the lowest at 1.4 g cm-3, the maximum physical compaction of the andosol in the compactor, and the difference in the Ct values between compacted and non-compacted soil was four cycles at the largest, suggesting that 16 times more DNA derived from SCN was detected by the compaction. Then, different numbers (10 to 3,000) of SCN eggs were added to 20 g of a non-infested andosol and the soils were compacted to 1.4 g cm-3. There was a significant correlation (r2 = 0.8615, P Ct values and the number of eggs added. These results demonstrated that the present method using a combination of soil compaction and real-time PCR enabled rapid and sensitive quantification of SCN eggs in soil.

Soybean cyst nematode (Heterodera glycines Ichinohe) is one of the serious soybean [Glycine max (L.) Merr.] pests in major soybean producing countries. The objective of this study was to investigate of Heterodera glycines type using the five SCN infested soybean field soils and was to evaluate resistance to the soybean cyst nematode HG 2.5.7 type on soybean varieties and germplasms. The five SCN contaminated soil samples were collected from the three provinces on November 2011 in Korea, and eggs were cultured on early spring season in 2012. For the second study, a total fifty nine soybean varieties and germplasms were tested by infestation of HG type 2.5.7 in the greenhouse. Soybean cyst nematode HG types were investigated from five locations, HG 2 (race 1) type at Donghae, HG 2.5 (race 1) type at Jeongseon and Hapcheon, HG type 2.5.7 (race 1 or 5) at Yeongwol, and HG 1.2.7 (race 5) type at Haenam locations in present study. No Korean soybean varieties and germplasms were observed with SCN resistant trait to the HG type 2.5.7. Average SCN female index were calculated with 82.7% in 59 plant materials. Our results could be provided useful information to develop a SCN resistant cultivar in Korea.

Ligand mimicry? Plant-parasitic nematode polypeptide with similarity to CLAVATA3

Spread of soybean cyst nematode Heterodera glycines (SCN) to much of the soybean (Glycine max) growing region in the Midwest has created a persistent and significant annual yield loss for soybean. Host resistance has been the primary means of reducing yield loss to SCN. It is not known how moderately resistant cultivars fit into the management of SCN. Moderately resistant cultivars can have high yield potential, but nematode reproduction is greater than on resistant cultivars. Moderate resistance is defined by a SCN female index (FI) of 10 to 29 in standardized tests, whereas cultivars with an FI &lt; 10 are considered resistant. Two each of SCN-resistant, moderately resistant, and susceptible (FI &gt; 60) cultivars were planted in the same plots for two soybean crops in annual rotation with corn. The SCN population was reduced 80 and 54% by resistant and moderately resistant cultivars, respectively, and increased 189% by the susceptible. Yields of the resistant and moderately resistant were 8.2 and 11.8 bu/acre better, respectively, than for the susceptible. All plots were planted to a susceptible cultivar in the final year of the study, and demonstrated there was a carry-over effect from previous cultivars. Following resistant and moderately resistant cultivars, yields of the susceptible were 6.6 and 4.3 bu/acre above following susceptible cultivars. This study showed that moderately resistant soybean cultivars can be an effective tool for improving profitability of soybean. Accepted for publication 9 April 2008. Published 18 June 2008.

Soybean cyst nematode Heterodera glycines (SCN) is a major threat to global soybean production. Effective management of this disease is dependent on the development of resistant cultivars. Two SCN HG Types, 7 and 1.3.4.7. were previously identified as prevalent H. glycines populations in Northeast China. In order to evaluate soybean cultivars resistant to local SCN populations, 110 domestic commercial soybeans from different regions of Northeast China were assessed in the greenhouse to determine their potential as novel sources of resistance. The results suggested that cultivars responded differently to the two HG types. Of the 110 soybean cultivars evaluated, 24 accessions were classified as resistant or moderately resistant to HG Type 7, and five cultivars were classified as resistant or moderately resistant to HG Type 1.3.4.7. Among the tested cultivars, Kangxian 12 and Qingdou 13 had resistance response to both HG types 7 and 1.3.4.7. Thus, these broad-based SCN cultivars will be the valuable materials in the SCN resistance breeding program.

Soybean cyst nematode (Heterodera glycines Ichinohe), a devastating pathogen in soybean, was chosen as a model system to investigate nematode behavior and gene expression changes in response to acidic and basic pH and salt signals (pH 4.5, 5.25, 8.6, and 10 and NaCl) through full-length transcriptome sequencing of 18 samples. An average of 4.36 Gbp of clean reads per sample were generated, and 3972 novel genes and 29,529 novel transcripts were identified. Sequence structural variation during or after transcription may be associated with the nematode's behavioral response. The functional analysis of 1817/4962 differentially expressed genes/transcripts showed that signal transduction pathways, including transmembrane receptors, ion channels, and Ca2+ transporters, were activated, but pathways involved in nematode development (e.g., ribosome) and energy production (e.g., oxidative phosphorylation) were inhibited. A corresponding model was established. Our findings suggest that these receptors and ion channels might be potential targets for nematicides or drug discovery.

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant-parasitic cyst nematodes secrete CLE-like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode-induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock-down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.

Plant parasitic nematodes respond to root exudates to locate their host roots. In our studies second stage juveniles of Heterodera glycines, the soybean cyst nematode (SCN), quickly migrated to soybean roots in Pluronic F-127 gel. Roots of soybean and non-host Arabidopsis treated with the ethylene (ET)-synthesis inhibitor aminoethoxyvinylglycine (AVG) were more attractive to SCN than untreated roots, and significantly more nematodes penetrated into roots. Moreover, Arabidopsis ET insensitive mutants (ein2, ein2-1, ein2-5, ein3-1, ein5-1, and ein6) were more attractive than wild-type plants. Conversely, the constitutive triple-response mutant ctr1-1, was less attractive to SCN. While ET receptor gain-of-function mutant ein4-1 attracted more SCN than the wild-type, there were no significant differences in attractiveness between another gain-of-function ET receptor mutant, etr1-3, or the loss-of-function mutants etr1-7 and ers1-3 and the wild type. Expression of the reporter construct EBS: β-glucuronidase (GUS) was detected in Arabidopsis root tips as early as 6 h post infection, indicating that ET signaling was activated in Arabidopsis early by SCN infection. These results suggest that an active ET signaling pathway reduces root attractiveness to SCN in a way similar to that reported for root-knot nematodes, but opposite to that suggested for the sugar beet cyst nematode Heterodera schachtii.

Cyst nematodes are sedentary plant parasites that cause dramatic cellular changes in the plant root to form feeding cells, so-called syncytia. 10A06 is a cyst nematode secretory protein that is most likely secreted as an effector into the developing syncytia during early plant parasitism. A homolog of the uncharacterized soybean cyst nematode (Heterodera glycines), 10A06 gene was cloned from the sugar beet cyst nematode (Heterodera schachtii), which is able to infect Arabidopsis (Arabidopsis thaliana). Constitutive expression of 10A06 in Arabidopsis affected plant morphology and increased susceptibility to H. schachtii as well as to other plant pathogens. Using yeast two-hybrid assays, we identified Spermidine Synthase2 (SPDS2), a key enzyme involved in polyamine biosynthesis, as a specific 10A06 interactor. In support of this protein-protein interaction, transgenic plants expressing 10A06 exhibited elevated SPDS2 mRNA abundance, significantly higher spermidine content, and increased polyamine oxidase (PAO) activity. Furthermore, the SPDS2 promoter was strongly activated in the nematode-induced syncytia, and transgenic plants overexpressing SPDS2 showed enhanced plant susceptibility to H. schachtii. In addition, in planta expression of 10A06 or SPDS2 increased mRNA abundance of a set of antioxidant genes upon nematode infection. These data lend strong support to a model in which the cyst nematode effector 10A06 exerts its function through the interaction with SPDS2, thereby increasing spermidine content and subsequently PAO activity. Increasing PAO activity results in stimulating the induction of the cellular antioxidant machinery in syncytia. Furthermore, we observed an apparent disruption of salicylic acid defense signaling as a function of 10A06. Most likely, increased antioxidant protection and interruption of salicylic acid signaling are key aspects of 10A06 function in addition to other physiological and morphological changes caused by altered polyamines, which are potent plant signaling molecules.

The soybean cyst nematode (SCN) Heterodera glycines continues to be a major threat to soybean production worldwide. Morphological discrimination between SCN and other nematodes of the Heterodera schachtii sensu stricto group is not only difficult and time-consuming but also requires high expertise in nematode taxonomy. Molecular assays were developed to differentiate SCN from sugar beet cyst nematode (SBCN) and other nematodes and to quantify SCN directly from DNA extracts of field soils. SCN- and SBCN-specific quantitative real-time PCR (qPCR) primers were designed from a nematode-secreted CLAVATA gene and used for these assays. The primers were evaluated on the basis of target specificity to SCN or SBCN using DNA from 20 isolates of SCN and 32 isolates of other plant-parasitic nematodes. A standard curve relating threshold cycle and log values of nematode numbers was generated from artificially infested soils and was used to quantify SCN in naturally infested field soils. There was a high correlation between the SCN numbers estimated from naturally infested field soils by conventional methods, and the numbers quantified using the SYBR Green I-based qPCR assay. The qPCR assay is highly specific and sensitive and provides improved SCN detection sensitivity down to 1 SCN egg in 20 g of soil (10 eggs/200 g soil). This assay is useful for efficient detection and quantification of SCN directly from field soil. Species-specific conventional PCR assays were also developed each for SCN and SBCN, alongside a qPCR assay that simultaneously discriminates SCN from SBCN. These assays require no expertise in nematode taxonomy and morphology, and they may serve as useful diagnostic tools in research, diagnostic laboratories, and extension services for SCN management. Sensitive and accurate detection and quantification of SCN are essential for recommending effective management measures against SCN. We also investigated the impact of soil texture and nematode life stage on molecular quantification of SCN.